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Background and Purpose: In mouse tail arteries, selective alpha(2)-adrenoceptor antagonism with rauwolscine caused powerful dilation during constriction to the alpha(1)-adrenoceptor agonist phenylephrine. This study therefore assessed phenylephrine's selectivity at vascular alpha-adrenoceptors and the mechanism(s) underlying dilation to rauwolscine.Experimental Approach: Mouse isolated tail arteries were assessed using a pressure myograph.Key Results: The alpha(2)-adrenoceptor agonist UK14,304 caused low-maximum constriction that was inhibited by rauwolscine (3 x 10(-8) M) but not by the selective alpha(1)-adrenoceptor antagonist prazosin (10(-7) M). Concentration-effect curves to phenylephrine, cirazoline or noradrenaline were unaffected by rauwolscine but were inhibited by prazosin, which was more effective at high compared with low levels of constriction. In the presence of prazosin, rauwolscine inhibited the curves and was more effective at low compared with high levels of constriction. Although rauwolscine alone did not affect concentration-effect curves to phenylephrine, noradrenaline or cirazoline, it caused marked transient dilation when administered during constriction to these agonists. Dilation was mimicked by another alpha(2)-adrenoceptor antagonist (RX821002, 3 x 10(-8) M), was dependent on agonist selectivity, and did not occur during adrenoceptor-independent constriction (U46619). During constriction to UK14,304 plus U46619, rauwolscine or rapid removal of UK14,304 caused transient dilation that virtually abolished the combined constriction. Endothelial denudation reduced these dilator responses.Conclusions and Implications: Inhibition of alpha(2)-adrenoceptors caused transient dilation that was substantially greater than the contribution of alpha(2)-adrenoceptors to the constriction. This reflects a slowly reversing alpha(2)-adrenoceptor-mediated endothelium-dependent dilation and provides a rapid, sensitive test of alpha(2)-adrenoceptor activity. This approach also clearly emphasizes the poor selectivity of phenylephrine at vascular alpha-adrenoceptors. [ABSTRACT FROM AUTHOR]

Cooling causes cutaneous dilatation to restrain cold-induced constriction and prevent tissue injury. Cooling increases communication through myoendothelial gap junctions (MEGJs), thereby increasing endothelium-derived hyperpolarization (EDH)-type dilatation. EDH is initiated by calcium-activated potassium channels (K Ca ) activated by endothelial stimuli or muscle-derived mediators traversing MEGJs (myoendothelial feedback). The goal of this study was to determine the individual roles of K Ca with small (SK3) and intermediate (IK1) conductance in cooling-induced dilatation. Vasomotor responses of mice isolated cutaneous tail arteries were analyzed by pressure myography at 37°C and 28°C. Cooling increased acetylcholine-induced EDH-type dilatation during inhibition of NO and prostacyclin production. IK1 inhibition did not affect dilatations to acetylcholine, whereas SK3 inhibition inhibited dilatation at both temperatures. Cooling uncovered myoendothelial feedback to inhibit constrictions in U46619. IK1 inhibition did not affect U46619 constrictions, whereas SK3 inhibition abolished the inhibitory effect of cooling without affecting U46619 constriction at 37°C. Immunoblots confirmed SK3 expression, which was localized (immunofluorescence) to holes in the internal elastic lamina consistent with myoendothelial projections. Immunoblots and Immunofluorescence did not detect IK1. Studies in non-cutaneous arteries have highlighted the predominant role of IK1 in EDH-type dilatation. Cutaneous arteries are distinctly reliant on SK3, which may enable EDH-type dilation to be amplified by cooling., (© 2023 The Authors. Physiological Reports published by Wiley Periodicals LLC on behalf of The Physiological Society and the American Physiological Society.)

Purpose of Review: Utilizing recent insight into the vasculopathy of scleroderma (SSc), the review will highlight new opportunities for evaluating and treating the disease by promoting stabilization and protection of the microvasculature., Recent Findings: Endothelial junctional signaling initiated by vascular endothelial-cadherin (VE-cadherin) and Tie2 receptors, which are fundamental to promoting vascular health and stability, are disrupted in SSc. This would be expected to not only diminish their protective activity, but also increase pathological processes that are normally restrained by these signaling mediators, resulting in pathological changes in vascular function and structure. Indeed, key features of SSc vasculopathy, from the earliest signs of edema and puffy fingers to pathological disruption of hemodynamics, nutritional blood flow, capillary structure and angiogenesis are all consistent with this altered endothelial signaling. It also likely contributes to further progression of the disease including tissue fibrosis, and organ and tissue injury., Summary: Restoring protective endothelial junctional signaling should combat the vasculopathy of SSc and prevent further deterioration in vascular and organ function. Indeed, this type of targeted approach has achieved remarkable results in preclinical models for other diseases. Furthermore, tracking this endothelial junctional signaling, for example by assessing vascular permeability, should facilitate insight into disease progression and its response to therapy., (Copyright © 2021 Wolters Kluwer Health, Inc. All rights reserved.)

Objective: Intravenous iloprost improves Raynaud's phenomenon (RP) and promotes healing of digital ulcers in systemic sclerosis (SSc; scleroderma). Despite a short half-life, its clinical efficacy lasts weeks. Endothelial adherens junctions, which are formed by VE-cadherin clustering between endothelial cells (ECs), regulate endothelial properties including barrier function, endothelial-to-mesenchymal transition (EndoMT), and angiogenesis. We undertook this study to investigate the hypothesis that junctional disruption contributes to vascular dysfunction in SSc, and that the protective effect of iloprost is mediated by strengthening of those junctions., Methods: Dermal ECs from SSc patients and healthy controls were isolated. The effect of iloprost on ECs was examined using immunofluorescence, permeability assays, Matrigel tube formation, and quantitative polymerase chain reaction., Results: Adherens junctions in SSc were disrupted compared to normal ECs, as indicated by reduced levels of VE-cadherin and increased permeability in SSc ECs (P < 0.05). Iloprost increased VE-cadherin clustering at junctions and restored junctional levels of VE-cadherin in SSc ECs (mean ± SD 37.3 ± 4.3 fluorescence units) compared to normal ECs (mean ± SD 29.7 ± 3.4 fluorescence units; P < 0.05), after 2 hours of iloprost incubation. In addition, iloprost reduced permeability of monolayers, increased tubulogenesis, and blocked EndoMT in both normal and SSc ECs (n ≥ 3; P < 0.05). The effects in normal ECs were inhibited by a function-blocking antibody that prevents junctional clustering of VE-cadherin., Conclusion: Our data suggest that the long-lasting effects of iloprost reflect its ability to stabilize adherens junctions, resulting in increased tubulogenesis and barrier function and reduced EndoMT. These findings provide a mechanistic basis for the use of iloprost in treating SSc patients with RP and digital ulcers., (© 2020, American College of Rheumatology.)

Cold exposure causes cutaneous vasoconstriction via a reflex increase in sympathetic activity and a local effect to augment adrenergic constriction. Local cooling also initiates cutaneous dilatation, which may function to restrain cold-induced constriction. However, the underlying mechanisms and physiological role of cold-induced dilatation have not been defined. Experiments were performed to assess the role of endothelial-derived mediators in this response. In isolated pressurized cutaneous mouse tail arteries, cooling (28°C) did not affect the magnitude of dilatation to acetylcholine in preconstricted arteries. However, inhibition of nitric oxide (NO) [ N G -nitro-l-arginine methyl ester (l-NAME)] and prostacyclin (PGI 2 ) (indomethacin) reduced acetylcholine-induced dilatation at 37°C but not at 28°C, suggesting that cooling increased NO/PGI 2 -independent dilatation. This NO/PGI 2 -independent dilatation was reduced by inhibition of endothelial SK (UCL1684) and IK (TRAM34) Ca 2+ -activated K + -channels (K Ca ), consistent with endothelium-derived hyperpolarization (EDH). Cooling also increased dilatation to direct activation of K Ca channels (SKA31, CyPPA) but did not affect dilatation to exogenous NO (DEA-NONOate). This cooling-induced increase in EDH-type dilatations was associated with divergent effects on potential downstream EDH mechanisms: cooling reduced dilatation to K + , which mimics an intercellular K + cloud, but increased direct communication between endothelial and smooth muscle cells (myoendothelial coupling), assessed by cellular transfer of biocytin. Indeed, inhibition of gap junctions (carbenoxolone) abolished the EDH-type component of dilatation to acetylcholine during cooling but did affect NO-dominated dilatation at 37°C. Cooling also inhibited U46619 constriction that was prevented by inhibition of IK and SK K Ca channels or inhibition of gap junctions. The results suggest that cooling dilates cutaneous arteries by increasing myoendothelial communication and amplifying EDH-type dilatation. NEW & NOTEWORTHY Cold causes cutaneous vasoconstriction to restrict heat loss. Although cold also initiates cutaneous dilatation, the mechanisms and role of this dilatation have not been clearly defined. This study demonstrates that cooling increases myoendothelial coupling between smooth muscle and endothelial cells in cutaneous arteries, which is associated with increased endothelium-derived hyperpolarization (EDH)-type dilatation. Dysfunction in this process may contribute to excessive cold-induced constriction and tissue injury.

Homodimer formation is essential for the normal activity of endothelial nitric oxide synthase (eNOS). Structural uncoupling of eNOS, with generation of enzyme monomers, is thought to contribute to endothelial dysfunction in several vascular disorders, including aging. However, low-temperature SDS-PAGE of healthy arteries has revealed considerable variation between studies in the relative expression of eNOS dimers and monomers. While assessing structural uncoupling of eNOS in aging arteries, we identified methodological pitfalls that might contribute to such variation. Therefore, using human cultured aortic endothelial cells and aortas from young and aged Fischer-344 rats, we investigated optimal approaches for analyzing the expression of eNOS monomers and dimers. The results demonstrated that published differences in treatment of cell lysates can significantly impact the relative expression of several eNOS species, including denatured monomers, partially folded monomers, dimers, and higher-order oligomers. In aortas, experiments initially confirmed a large increase in eNOS monomers in aging arteries, consistent with structural uncoupling. However, these monomers were actually endogenous IgG, which, under these conditions, has mobility similar to eNOS monomers. Increased IgG levels in aged aortas likely reflect the aging-induced disruption of endothelial junctions and increased arterial penetration of IgG. After removal of the IgG signal, there were low levels of eNOS monomers in young arteries, which were not significantly different in aged arteries. Therefore, structural uncoupling of eNOS is not a prominent feature in young healthy arteries, and the process is not increased by aging. The study also identifies optimal approaches to analyze eNOS dimers and monomers. NEW & NOTEWORTHY Structural uncoupling of endothelial nitric oxide synthase (eNOS) is considered central to endothelial dysfunction. However, reported levels of eNOS dimers and monomers vary widely, even in healthy arteries. We demonstrate that sample processing can alter relative levels of eNOS species. Moreover, endothelial dysfunction in aging aortas results in IgG accumulation, which, because of similar mobility to eNOS monomers, could be misinterpreted as structural uncoupling. Indeed, enzyme monomerization is not prominent in young or aging arteries.

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 Tyr 416 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.

Key Points: Ageing-induced endothelial dysfunction contributes to organ dysfunction and progression of cardiovascular disease. VE-cadherin clustering at adherens junctions promotes protective endothelial functions, including endothelium-dependent dilatation. Ageing increased internalization and degradation of VE-cadherin, resulting in impaired activity of adherens junctions. Inhibition of VE-cadherin clustering at adherens junctions (function-blocking antibody; FBA) reduced endothelial dilatation in young arteries but did not affect the already impaired dilatation in old arteries. After junctional disruption with the FBA, dilatation was similar in young and old arteries. Src tyrosine kinase activity and tyrosine phosphorylation of VE-cadherin were increased in old arteries. Src inhibition increased VE-cadherin at adherens junctions and increased endothelial dilatation in old, but not young, arteries. Src inhibition did not increase dilatation in old arteries treated with the VE-cadherin FBA. Ageing impairs the activity of adherens junctions, which contributes to endothelial dilator dysfunction. Restoring the activity of adherens junctions could be of therapeutic benefit in vascular ageing., Abstract: Endothelial dilator dysfunction contributes to pathological vascular ageing. Experiments assessed whether altered activity of endothelial adherens junctions (AJs) might contribute to this dysfunction. Aortas and tail arteries were isolated from young (3-4 months) and old (22-24 months) F344 rats. VE-cadherin immunofluorescent staining at endothelial AJs and AJ width were reduced in old compared to young arteries. A 140 kDa VE-cadherin species was present on the cell surface and in TTX-insoluble fractions, consistent with junctional localization. Levels of the 140 kDa VE-cadherin were decreased, whereas levels of a TTX-soluble 115 kDa VE-cadherin species were increased in old compared to young arteries. Acetylcholine caused endothelium-dependent dilatation that was decreased in old compared to young arteries. Disruption of VE-cadherin clustering at AJs (function-blocking antibody, FBA) inhibited dilatation to acetylcholine in young, but not old, arteries. After the FBA, there was no longer any difference in dilatation between old and young arteries. Src activity and tyrosine phosphorylation of VE-cadherin were increased in old compared to young arteries. In old arteries, Src inhibition (saracatinib) increased: (i) 140 kDa VE-cadherin in the TTX-insoluble fraction, (ii) VE-cadherin intensity at AJs, (iii) AJ width, and (iv) acetylcholine dilatation. In old arteries treated with the FBA, saracatinib no longer increased acetylcholine dilatation. Saracatinib did not affect dilatation in young arteries. Therefore, ageing impairs AJ activity, which appears to reflect Src-induced phosphorylation, internalization and degradation of VE-cadherin. Moreover, impaired AJ activity can account for the endothelial dilator dysfunction in old arteries. Restoring endothelial AJ activity may be a novel therapeutic approach to vascular ageing., (© 2017 The Authors. The Journal of Physiology © 2017 The Physiological Society.)

Under physiological conditions, the arterial endothelium exerts a powerful protective influence to maintain vascular homeostasis. However, during the development of vascular disease, these protective activities are lost, and dysfunctional endothelial cells actually promote disease pathogenesis. Numerous investigations have analyzed the characteristics of dysfunctional endothelium with a view to understanding the processes responsible for the dysfunction and to determining their role in vascular pathology. This review adopts an alternate approach: reviewing the mechanisms that contribute to the initial formation of a healthy protective endothelium and on how those mechanisms may be disrupted, precipitating the appearance of dysfunctional endothelial cells and the progression of vascular disease. This approach, which highlights the role of endothelial adherens junctions and vascular endothelial-cadherin in endothelial maturation and endothelial dysfunction, provides new insight into the remarkable biology of this important cell layer and its role in vascular protection and vascular disease.

Short chain fatty acid (SCFA) metabolites are byproducts of gut microbial metabolism that are known to affect host physiology via host G protein-coupled receptor (GPCRs). We previously showed that an acute SCFA bolus decreases blood pressure (BP) in anesthetized mice, an effect mediated primarily via Gpr41. In this study, our aims were to identify the cellular localization of Gpr41 and to determine its role in BP regulation. We localized Gpr41 to the vascular endothelium using RT-PCR: Gpr41 is detected in intact vessels (with endothelium) but is absent from denuded vessels (without endothelium). Furthermore, using pressure myography we confirmed that SCFAs dilate resistance vessels in an endothelium-dependent manner. Since we previously found that Gpr41 mediates a hypotensive response to acute SCFA administration, we hypothesized that Gpr41 knockout (KO) mice would be hypertensive. Here, we report that Gpr41 KO mice have isolated systolic hypertension compared with wild-type (WT) mice; diastolic BP was not different between WT and KO. Older Gpr41 KO mice also exhibited elevated pulse wave velocity, consistent with a phenotype of systolic hypertension; however, there was no increase in ex vivo aorta stiffness (measured by mechanical tensile testing). Plasma renin concentrations were also similar in KO and WT mice. The systolic hypertension in Gpr41 KO is not salt sensitive, as it is not significantly altered on either a high- or low-salt diet. In sum, these studies suggest that endothelial Gpr41 lowers baseline BP, likely by decreasing active vascular tone without altering passive characteristics of the blood vessels, and that Gpr41 KO mice have hypertension of a vascular origin., (Copyright © 2016 the American Physiological Society.)

Key Points: Endothelial expression and the release of endothelin-1 (ET-1) in levels sufficient to initiate vasoconstriction is considered to be a hallmark feature of pathological endothelial dysfunction. During the immediate postnatal period, arterial endothelial cells undergo remarkable structural and functional changes as they transition to a mature protective cell layer, which includes a marked increase in NO dilator activity. The present study demonstrates that endothelial cells lining newborn central arteries express high levels of ET-1 peptides and, in response to endothelial stimulation, rapidly release ET-1 and initiate powerful ET-1-mediated constriction. This activity is lost as the endothelium matures in the postnatal period. Heightened activity of ET-1 in the neonatal endothelium might contribute to inappropriate responses of immature arteries to stress or injury. Indeed, the immature endothelium resembles dysfunctional endothelial cells, and retention or re-emergence of this phenotype may contribute to the development of vascular disease., Abstract: Endothelial cells lining fetal and newborn arteries have an unusual phenotype, including reduced NO activity, prominent actin stress fibres and poorly developed cellular junctions. Experiments were performed to determine whether the immature endothelium of newborn arteries also expresses and releases endothelin-1 (ET-1) and initiates endothelium-dependent constriction. Carotid arteries were isolated from newborn (postnatal day 1; P1), postnatal day 7 (P7) and postnatal day 21 (P21) mice and assessed in a pressure myograph system. Endothelial stimulation with A23187 or thrombin caused constriction in P1 arteries, no significant change in diameter of P7 arteries, and dilatation in P21 arteries. In P1 arteries, constriction to thrombin or A23187 was inhibited by endothelial-denudation, by ET-1 receptor antagonists (BQ123 plus BQ788) or by inhibition of endothelin-converting enzyme (phosphoramidon or SM19712). ET-1 receptor antagonism did not affect responses to thrombin or A23187 in more mature arteries. Exogenous ET-1 caused similar concentration-dependent constrictions of P1, P7 and P21 arteries. Endothelial stimulation with thrombin rapidly increased the endothelial release of ET-1 from P1 but not P21 aortas. Endothelial expression of ET-1 peptides, as assessed by immunofluorescence analysis, was increased in P1 compared to P21 arteries. Therefore, newborn endothelial cells express high levels of ET-1 peptides, rapidly release ET-1 in response to endothelial stimulation, and initiate ET-1-mediated endothelium-dependent constriction. This activity is diminished as the endothelium matures in the immediate postnatal period. Heightened activity of ET-1 in neonatal endothelium probably reflects an early developmental role of the peptide, although this might contribute to inappropriate responses of immature arteries to stress or injury., (© 2016 The Authors. The Journal of Physiology © 2016 The Physiological Society.)

Aging impairs endothelium-dependent NO-mediated dilatation, which results from increased production of reactive oxygen species (ROS). The local generation of angiotensin II (ANG II) is increased in aging arteries and contributes to inflammatory and fibrotic activity of smooth muscle cells and arterial wall remodeling. Although prolonged in vivo ANG II inhibition improves the impaired endothelial dilatation of aging arteries, it is unclear whether this reflects inhibition of intravascular or systemic ANG II systems. Experiments were therefore performed on isolated tail arteries from young (3-4 mo) and old (22-24 mo) F344 rats to determine if a local renin-angiotensin system contributes to the endothelial dilator dysfunction of aging. Aging impaired dilatation to the endothelial agonist acetylcholine but did not influence responses to a nitric oxide (NO) donor (DEA NONOate). Dilatation to acetylcholine was greatly reduced by NO synthase inhibition [nitro-l-arginine methyl ester (l-NAME)] in young and old arteries. In isolated arteries, acute inhibition of angiotensin-converting enzyme (ACE) (perindoprilat), renin (aliskiren), or AT1 receptors (valsartan, losartan) did not influence dilatation to acetylcholine in young arteries but increased responses in old arteries. After ANG II inhibition, the dilator response to acetylcholine was similar in young and old arteries. ROS activity, which was increased in endothelium of aging arteries, was also reduced by inhibiting ANG II (perindoprilat, losartan). Renin expression was increased by 5.6 fold and immunofluorescent levels of ANG II were confirmed to be increased in aging compared with young arteries. Exogenous ANG II inhibited acetylcholine-induced dilatation. Therefore, aging-induced impairment of endothelium-dependent dilatation in aging is caused by a local intravascular renin-angiotensin system., (Copyright © 2016 the American Physiological Society.)

During exposure to cold, our bodies attempt to maintain normal core temperature by restricting heat loss through cutaneous vasoconstriction, and by increasing heat production through shivering and nonshivering thermogenesis. In selected areas of human skin (including on the fingers and toes), the vascular system has specialized structural and functional features that enable it to contribute to thermoregulation. These features include arteriovenous anastomoses, which directly connect the arterial and venous systems and bypass the nutritional capillaries supplying blood to the skin tissue. Of note, Raynaud phenomenon predominantly affects the arterial territories supplying these specialized areas of skin. Indeed, Raynaud phenomenon can be considered a disorder of vascular thermoregulatory control. This Review presents an understanding of Raynaud phenomenon in the context of vascular and thermoregulatory control mechanisms, including the role of unique thermosensitive vascular structural and functional specialization, and describes the potential role of thermogenesis in this disorder. This new approach provides remarkable insight into the disease process and builds a framework to critically appraise the existing knowledge base. This paradigm also explains the deficiencies in some current therapeutic approaches, and highlights new areas of potential relevance to the pathogenesis and treatment of Raynaud phenomenon that should be expanded and explored.

Experiments were performed to determine whether or not acute exposure to elevated pressure would disrupt endothelium-dependent dilatation by increasing local angiotensin II (ANG II) signaling. Vasomotor responses of mouse-isolated carotid arteries were analyzed in a pressure myograph at a control transmural pressure (PTM) of 80 mmHg. Acetylcholine-induced dilatation was reduced by endothelial denudation or by inhibition of nitric oxide synthase (NG-nitro-L-arginine methyl ester, 100 μM). Transient exposure to elevated PTM (150 mmHg, 180 min) inhibited dilatation to acetylcholine but did not affect responses to the nitric oxide donor diethylamine NONOate. Elevated PTM also increased endothelial reactive oxygen species, and the pressure-induced endothelial dysfunction was prevented by the direct antioxidant and NADPH oxidase inhibitor apocynin (100 μM). The increase in endothelial reactive oxygen species in response to elevated PTM was reduced by the ANG II type 1 receptor (AT1R) antagonists losartan (3 μM) or valsartan (1 μM). Indeed, elevated PTM caused marked expression of angiotensinogen, the precursor of ANG II. Inhibition of ANG II signaling, by blocking angiotensin-converting enzyme (1 μM perindoprilat or 10 μM captopril) or blocking AT1Rs prevented the impaired response to acetylcholine in arteries exposed to 150 mmHg but did not affect dilatation to the muscarinic agonist in arteries maintained at 80 mmHg. After the inhibition of ANG II, elevated pressure no longer impaired endothelial dilatation. In arteries treated with perindoprilat to inhibit endogenous formation of the peptide, exogenous ANG II (0.3 μM, 180 min) inhibited dilatation to acetylcholine. Therefore, elevated pressure rapidly impairs endothelium-dependent dilatation by causing ANG expression and enabling ANG II-dependent activation of AT1Rs. These processes may contribute to the pathogenesis of hypertension-induced vascular dysfunction and organ injury., (Copyright © 2015 the American Physiological Society.)

Endothelium of fetal or newborn arteries is atypical, displaying actin stress fibers and reduced nitric oxide (NO)-mediated dilatation. This study tested the hypothesis that Rho/Rho kinase signaling, which promotes endothelial stress fibers and inhibits endothelial dilatation, contributed to this phenotype. Carotid arteries were isolated from newborn [postnatal day 1 (P1)], P7, and P21 mice. Endothelial dilatation to acetylcholine (pressure myograph) was minimal at P1, increased at P7, and further increased at P21. Inhibition of Rho (C3 transferase) or Rho kinase (Y27632, fasudil) significantly increased dilatation to acetylcholine in P1 arteries but had no effect in P7 or P21 arteries. After inhibition of NO synthase (N(G)-nitro-l-arginine methyl ester), Rho kinase inhibition no longer increased acetylcholine responses in P1 arteries. Rho kinase inhibition did not affect dilatation to the NO donor DEA-NONOate. The endothelial actin cytoskeleton was labeled with phalloidin and visualized by laser-scanning microscopy. In P1 arteries, the endothelium had prominent transcytoplasmic stress fibers, whereas in P7 and P21 arteries, the actin fibers had a significantly reduced intensity and were restricted to cell borders. Phosphorylation of myosin light chains, a Rho kinase substrate, was highest in P1 endothelium and significantly reduced in P7 and P21 endothelium (laser-scanning microscopy). In P1 arteries, inhibition of Rho (C3 transferase) or Rho kinase (Y27632) significantly reduced the intensity of actin fibers, which were restricted to cell borders. Similarly, in P1 arteries, Rho inhibition significantly reduced endothelial levels of phosphorylated myosin light chains. These results indicate that the atypical function and morphology of newborn endothelium is mediated by Rho/Rho kinase signaling., (Copyright © 2014 the American Physiological Society.)

Background: Vascular aging is closely associated with increased vascular stiffness. It has recently been demonstrated that decreased nitric oxide (NO)-induced S-nitrosylation of tissue transglutaminase (TG2) contributes to age-related vascular stiffness. In the current study, we tested the hypothesis that exercise restores NO signaling and attenuates vascular stiffness by decreasing TG2 activity and cross-linking in an aging rat model., Methods and Results: Rats were subjected to 12 weeks of moderate aerobic exercise. Aging was associated with diminished phosphorylated endothelial nitric oxide synthase and phosphorylated vasodilator-stimulated phosphoprotein abundance, suggesting reduced NO signaling. TG2 cross-linking activity was significantly increased in old animals, whereas TG2 abundance remained unchanged. These alterations were attenuated in the exercise cohort. Simultaneous measurement of blood pressure and pulse wave velocity (PWV) demonstrated increased aortic stiffness in old rats, compared to young, at all values of mean arterial pressure (MAP). The PWV-MAP correlation in the old sedentary and old exercise cohorts was similar. Tensile testing of the vessels showed increased stiffness of the aorta in the old phenotype with a modest restoration of mechanical properties toward the young phenotype with exercise., Conclusions: Increased vascular stiffness during aging is associated with decreased TG2 S-nitrosylation, increased TG2 cross-linking activity, and increased vascular stiffness likely the result of decreased NO bioavailability. In this study, a brief period of moderate aerobic exercise enhanced NO signaling, attenuated TG cross-linking activity, and reduced ex vivo tensile properties, but failed to reverse functional vascular stiffness in vivo, as measured by PWV.

The second messenger cyclic AMP (cAMP) plays a vital role in vascular physiology, including vasodilation of large blood vessels. We recently demonstrated cAMP activation of Epac-Rap1A and RhoA-Rho-associated kinase (ROCK)-F-actin signaling in arteriolar-derived smooth muscle cells increases expression and cell surface translocation of functional α2C-adrenoceptors (α2C-ARs) that mediate vasoconstriction in small blood vessels (arterioles). The Ras-related small GTPAse Rap1A increased expression of α2C-ARs and also increased translocation of perinuclear α2C-ARs to intracellular F-actin and to the plasma membrane. This study examined the mechanism of translocation to better understand the role of these newly discovered mediators of blood flow control, potentially activated in peripheral vascular disorders. We utilized a yeast two-hybrid screen with human microvascular smooth muscle cells (microVSM) cDNA library and the α2C-AR COOH terminus to identify a novel interaction with the actin cross-linker filamin-2. Yeast α-galactosidase assays, site-directed mutagenesis, and coimmunoprecipitation experiments in heterologous human embryonic kidney (HEK) 293 cells and in human microVSM demonstrated that α2C-ARs, but not α2A-AR subtype, interacted with filamin. In Rap1-stimulated human microVSM, α2C-ARs colocalized with filamin on intracellular filaments and at the plasma membrane. Small interfering RNA-mediated knockdown of filamin-2 inhibited Rap1-induced redistribution of α2C-ARs to the cell surface and inhibited receptor function. The studies suggest that cAMP-Rap1-Rho-ROCK signaling facilitates receptor translocation and function via phosphorylation of filamin-2 Ser(2113). Together, these studies extend our previous findings to show that functional rescue of α2C-ARs is mediated through Rap1-filamin signaling. Perturbation of this signaling pathway may lead to alterations in α2C-AR trafficking and physiological function.

Experiments investigated maturation of endothelial function in the postnatal period. Carotid arteries isolated from newborn (postnatal day 1, P1) to P21 mice were assessed in myographs at transmural pressure (PTM) of 20 mmHg (P1 blood pressure, BP). Acetylcholine was ineffective in P1 but powerfully dilated P7 arteries, whereas NO-donor DEA-NONOate caused similar dilation at P1 and P7. Dilation to acetylcholine at P7 was abolished by inhibition of NO synthase (NOS) (l-NAME) or of phosphoinositide-3-kinase (PI3K) (wortmannin, LY294002). Endothelial NOS (eNOS) expression decreased in P7 compared with P1 arteries, although acetylcholine increased PO4-eNOS-Ser(1177) in P7 but not in P1 arteries. Endothelial maturation may therefore reflect increased signaling through PI3K, Akt, and eNOS. Systemic BP increases dramatically in the early postnatal period. After exposing P1 arteries to transient increased PTM (50 mmHg, 60 min), acetylcholine caused powerful dilation and increased PO4-eNOS-Ser(1177). Pressure-induced rescue of acetylcholine dilation was abolished by PI3K or NOS inhibition. Transient increased PTM did not affect dilation at P7, or dilation to NO-donor in P1 arteries. Width of endothelial adherens junctions (VE-cadherin immunofluorescence) increased significantly from P1 to P7, and in P1 arteries exposed to transient increased PTM. A function-blocking antibody to VE-cadherin reduced the pressure-induced rescue of acetylcholine responses at P1, and the dilation to acetylcholine in P7 arteries. Therefore, maturation of newborn endothelium dilator function may be induced by increasing BP in the postnatal period. Furthermore, this may be mediated by VE-cadherin signaling at adherens junctions. Interruption of this maturation pathway may contribute to developmental and adult vascular diseases.

The multifunctional enzyme tissue transglutaminase (TG2) contributes to the development and progression of several cardiovascular diseases. Extracellular rather than intracellular TG2 is enzymatically active, however, the mechanism by which it is exported out of the cell remains unknown. Nitric oxide (NO) is shown to constrain TG2 externalization in endothelial and fibroblast cells. Here, we examined the role of both exogenous and endogenous (endothelial cell-derived) NO in regulating TG2 localization in vascular cells and tissue. NO synthase inhibition in endothelial cells (ECs) using N-nitro L-arginine methyl ester (L-NAME) led to a time-dependent decrease in S-nitrosation and increase in externalization of TG2. Laminar shear stress led to decreased extracellular TG2 in ECs. S-nitrosoglutathione treatment led to decreased activity and externalization of TG2 in human aortic smooth muscle and fibroblast (IMR90) cells. Co-culture of these cells with ECs resulted in increased S-nitrosation and decreased externalization and activity of TG2, which was reversed by L-NAME. Aged Fischer 344 rats had higher tissue scaffold-associated TG2 compared to young. NO regulates intracellular versus extracellular TG2 localization in vascular cells and tissue, likely via S-nitrosation. This in part, explains increased TG2 externalization and activity in aging aorta.

Intracellular signaling by the second messenger cyclic AMP (cAMP) activates the Ras-related small GTPase Rap1 through the guanine exchange factor Epac. This activation leads to effector protein interactions, activation, and biological responses in the vasculature, including vasorelaxation. In vascular smooth muscle cells derived from human dermal arterioles (microVSM), Rap1 selectively regulates expression of G protein-coupled α(2C)-adrenoceptors (α(2C)-ARs) through JNK-c-jun nuclear signaling. The α(2C)-ARs are generally retained in the trans-Golgi compartment and mobilize to the cell surface and elicit vasoconstriction in response to cellular stress. The present study used human microVSM to examine the role of Rap1 in receptor localization. Complementary approaches included murine microVSM derived from tail arteries of C57BL6 mice that express functional α(2C)-ARs and mice deficient in Rap1A (Rap1A-null). In human microVSM, increasing intracellular cAMP by direct activation of adenylyl cyclase by forskolin (10 μM) or selectively activating Epac-Rap signaling by the cAMP analog 8-pCPT-2'-O-Me-cAMP (100 μM) activated RhoA, increased α(2C)-AR expression, and reorganized the actin cytoskeleton, increasing F-actin. The α(2C)-ARs mobilized from the perinuclear region to intracellular filamentous structures and to the plasma membrane. Similar results were obtained in murine wild-type microVSM, coupling Rap1-Rho-actin dynamics to receptor relocalization. This signaling was impaired in Rap1A-null murine microVSM and was rescued by delivery of constitutively active (CA) mutant of Rap1A. When tested in heterologous HEK293 cells, Rap1A-CA or Rho-kinase (ROCK-CA) caused translocation of functional α(2C)-ARs to the cell surface (~4- to 6-fold increase, respectively). Together, these studies support vascular bed-specific physiological role of Rap1 and suggest a role in vasoconstriction in microVSM.

Chronic hypoxia causes pulmonary hypertension with vascular remodeling, increase in vascular tone, and altered reactivity to agonists. These changes involve alterations in multiple Ca(2+) pathways in pulmonary arterial smooth muscle cells (PASMCs). We have previously shown that vanilloid (TRPV)- and melastatin-related transient receptor potential (TRPM) channels are expressed in pulmonary arteries (PAs). Here we found that TRPV4 was the only member of the TRPV and TRPM subfamilies upregulated in PAs of chronic hypoxic rats. The increase in TRPV4 expression occurred within 1 day of hypoxia exposure, indicative of an early hypoxic response. TRPV4 in PASMCs were found to be mechanosensitive. Osmo-mechanical stress imposed by hypotonic solution activated Ca(2+) transients; they were inhibited by TRPV4 specific short interfering RNA, the TRPV blocker ruthenium red, and the cytochrome P450 epoxygenase inhibitor N-(methylsulfonyl)-2-(2-propynyloxy)-benzenehexanamide. Consistent with TRPV4 upregulation, the Ca(2+) response induced by the TRPV4 agonist 4α-phorbol 12,13-didecanoate and hypotonicity was potentiated in hypoxic PASMCs. Moreover, a significant myogenic tone, sensitive to ruthenium red, was observed in pressurized endothelium denuded small PAs of hypoxic but not normoxic rats. The elevated basal intracellular Ca(2+) concentration in hypoxic PASMCs was also reduced by ruthenium red. In extension of these results, the development of pulmonary hypertension, right heart hypertrophy, and vascular remodeling was significantly delayed and suppressed in hypoxic trpv4(-/-) mice. These results suggest the novel concept that TRPV4 serves as a signal pathway crucial for the development of hypoxia-induced pulmonary hypertension. Its upregulation may provide a pathogenic feed-forward mechanism that promotes pulmonary hypertension via facilitated Ca(2+) influx, subsequently enhanced myogenic tone and vascular remodeling.

G-protein coupled receptors (GPCRs) are generally considered to function as cell surface signaling structures that respond to extracellular mediators, many of which do not readily access the cell's interior. Indeed, most GPCRs are preferentially targeted to the plasma membrane. However, some receptors, including α(2C)-Adrenoceptors, challenge conventional concepts of GPCR activity by being preferentially retained and localized within intracellular organelles. This review will address the issues associated with this unusual GPCR localization and discuss whether it represents a novel sub-cellular niche for GPCR signaling, whether these receptors are being stored for rapid deployment to the cell surface, or whether they represent immature or incomplete receptor systems., (2011 Elsevier B.V. All rights reserved.)

Mice deficient in Notch3 have defects in arterial vascular smooth muscle cell (VSMC) mechanosensitivity, including impaired myogenic responses and autoregulation, and inappropriate VMSC orientation. Experiments were performed to determine if Notch3 is activated by mechanical stimulation and contributes to mechanosensitive responses of VSMCs, including cell realignment. Cyclic, uniaxial stretch (10%, 1 Hz) of human VSMCs caused Notch3 activation, demonstrated by a stretch-induced increase in hairy and enhancer of split 1/hairy-related transcription factor-1 expression, translocation of Notch3 to the nucleus, and a decrease in the Notch3 extracellular domain. These effects were prevented by inhibiting the expression [small interfering (si)RNA] or proteolytic activation of Notch3 {N-(R)-[2-(hydroxyaminocarbonyl)methyl]-4-methylpentanoyl-l-naphthylalanyl-l-alanine-2-aminoethyl amide (TAPI-1; 50 μmol/l) to inhibit TNF-α-converting enzyme (TACE) or N-[N-(3,5-difluorophenacetyl-l-alanyl)]-S-phenylglycine t-butyl ester (DAPT; 20 μmol/l) to inhibit γ-secretase}. Stretch increased the activity of ROS within VSMCs, determined using dichlorodihydrofluorescein fluorescence. Catalase (1,200 U/ml), which degrades H₂O₂, inhibited the stretch-induced activation of Notch3, whereas in nonstretched cells, increasing H₂O₂ activity [H₂O₂ or manganese(III) tetrakis(1-methyl-4-pyridyl)porphyrin] caused activation of Notch3. Stretch increased the activity of TACE, which was prevented by catalase. Stretch-induced activation of p38 MAPK in VSMCs was inhibited either by catalase or by inhibiting Notch3 expression (siRNA). Stretch caused VSMCs to realign perpendicular to the direction of the mechanical stimulus, which was significantly inhibited by catalase or by inhibiting the expression (siRNA) or activation of Notch3 (TAPI-1 or DAPT). Therefore, cyclic uniaxial stretch activates Notch3 signaling through a ROS-mediated mechanism, and the presence of Notch3 is necessary for proper stretch-induced cell alignment in VSMCs. This mechanism may contribute to the physiological role of Notch3 in mediating developmental maturation of VSMCs.

Rationale: Obstructive sleep apnea (OSA) is a risk factor for cardiovascular disease. We hypothesized that patients with OSA and no cardiovascular disease have oxidant-related microcirculatory endothelial dysfunction., Objectives: To evaluate the microcirculation in OSA., Methods: This study included seven patients with OSA and seven age- and weight-matched control subjects (mean age, 38 yr; mean body mass index, 32.5 kg/m²). All participants were free of cardiovascular risk factors. Participants received measurement of brachial artery flow-mediated dilation and forearm subcutaneous biopsy. Patients underwent repeated tests 12 weeks after treatment. Microcirculatory endothelial cells were isolated, and immunohistochemistry staining for peroxynitrite in the microcirculation was performed., Measurements and Main Results: Flow-mediated dilation was lower in patients than in control subjects at baseline (mean ± SEM: 5.7 ± 0.5 vs. 9.5 ± 0.6; P = 0.02) and increased after treatment (5.7-7.3; change, 1.7 ± 0.6; P = 0.04). Microcirculatory peroxynitrite deposit was higher in patients compared with control subjects (44.0 ± 1.6 vs. 21.8 ± 1.9 stain density units; P < 0.001) and decreased after treatment from 44.0 to 30.5 stain density units (change, -13.5 ± 2.9; P = 0.009). In patients, transcription of endothelial nitric oxide synthase decreased from 5.2 to -1.3 after treatment (change, 6.5 ± 2.5; P = 0.05), and transcription of superoxide dismutase1 decreased from -4.0 to -12.3 after treatment (change, -8.3 ± 2.1; P = 0.01). These changes persisted after adjustment for weight and underlying severity of OSA., Conclusions: This is the first direct evaluation of the microcirculation in OSA. Patients with OSA with low cardiovascular risk status had increased oxidant production in the microcirculation and endothelial dysfunction, both of which improved with treatment. Endothelial nitric oxide synthase transcription decreased with treatment.

Rationale: Circulating levels of endothelin (ET)-1 and endogenous ET(A)-mediated constriction are increased in human aging. The mechanisms responsible are not known., Objective: Investigate the storage, release, and activity of ET-1 system in arteries from young and aged Fischer-344 rats., Methods and Results: After NO synthase inhibition (L-NAME), thrombin contracted aged arteries, which was inhibited by endothelial denudation, ET(A) receptor antagonism (BQ123), and ECE inhibition (phosphoramidon, SM19712) or by inhibiting exocytosis (TAT-NSF, N-ethylmaleimide-sensitive factor inhibitor). Thrombin did not cause endothelium-dependent contraction of young arteries. In aged but not young arteries, thrombin rapidly increased ET-1 release, which was abolished by endothelium denudation or TAT-NSF. L-NAME did not affect ET-1 release. ET-1 immunofluorescent staining was punctate and distinct from von Willebrand factor (VWF). VWF and ET-1 immunofluorescent intensity was similar in young and aged quiescent arteries. Thrombin rapidly increased ET-1 staining and decreased VWF staining in aged but had no effect in young aortas. After L-NAME, thrombin decreased VWF staining in young aortas. NO donor DEA-NONOate (1 to 100 nmol/L) reversed thrombin-induced exocytosis in young (VWF) but not aged L-NAME-treated aortas (VWF, ET-1). Expression of preproET-1 mRNA and ECE-1 mRNA were increased in aged compared to young endothelium. BigET-1 levels and contraction to exogenous BigET-1 (but not ET-1) were also increased in aged compared to young arteries., Conclusions: The stimulated exocytotic release of ET-1 is dramatically increased in aged endothelium. This reflects increased reactivity of exocytosis, increased expression and storage of ET-1 precursor peptides, and increased expression of ECE-1. Altered endothelial exocytosis of ET-1 and other mediators may contribute to cardiovascular pathology in aging.

Rationale: Although an age-related decrease in NO bioavailability contributes to vascular stiffness, the underlying molecular mechanisms remain incompletely understood. We hypothesize that NO constrains the activity of the matrix crosslinking enzyme tissue transglutaminase (TG2) via S-nitrosylation in young vessels, a process that is reversed in aging., Objective: We sought to determine whether endothelium-dependent NO regulates TG2 activity by S-nitrosylation and whether this contributes to age-related vascular stiffness., Methods and Results: We first demonstrate that NO suppresses activity and increases S-nitrosylation of TG2 in cellular models. Next, we show that nitric oxide synthase (NOS) inhibition leads to increased surface and extracellular matrix-associated TG2. We then demonstrate that endothelium-derived bioactive NO primarily mediates its effects through TG2, using TG2(-/-) mice chronically treated with the NOS inhibitor l-N(G)-nitroarginine methyl ester (L-NAME). We confirm that TG2 activity is modulated by endothelium-derived bioactive NO in young rat aorta. In aging rat aorta, although TG2 expression remains unaltered, its activity increases and S-nitrosylation decreases. Furthermore, TG2 inhibition decreases vascular stiffness in aging rats. Finally, TG2 activity and matrix crosslinks are augmented with age in human aorta, whereas abundance remains unchanged., Conclusions: Decreased S-nitrosylation of TG2 and increased TG activity lead to enhanced matrix crosslinking and contribute to vascular stiffening in aging. TG2 appears to be the member of the transglutaminase family primarily contributing to this phenotype. Inhibition of TG2 could thus represent a therapeutic target for age-associated vascular stiffness and isolated systolic hypertension.

Occupational exposure to hand-operated vibrating tools causes a spectrum of pathological changes in the vascular, neurological, and musculoskeletal systems described as the hand-arm vibration syndrome (HAVS). Experiments were performed to determine the effects of acute vibration on the function of digital arteries. Rats paws were exposed to a vibrating platform (4 h, 125 Hz, constant acceleration of 49 m/s(2) root mean squared), and digital artery function was assessed subsequently in vitro using a pressure myograph system. Constriction to phenylephrine or 5-hydroxytryptamine was reduced in digital arteries from vibrated paws. However, after endothelium denudation, constriction to the agonists was no longer impaired in vibrated arteries. Inhibition of nitric-oxide synthase (NOS) with N(omega)-nitro-l-arginine methyl ester (l-NAME) increased constriction to phenylephrine or 5-hydroxytryptamine in vibrated but not control arteries and abolished the vibration-induced depression in constrictor responses. However, nitric oxide (NO) activity, determined using the NO-sensitive probe 4-amino-5-methylamino-2', 7'-difluorofluorescein, was reduced in vibrated compared with control arteries. Endogenous levels of reactive oxygen species (ROS), determined using the ROS-sensitive probe 5-(and 6)-chloromethyl-2',7'-dichlorodihydro-fluorescein, were increased in vibrated compared with control arteries. The increased ROS levels were abolished by L-NAME or by catalase, which degrades extracellular hydrogen peroxide. Catalase also increased constriction to phenylephrine or 5-hydroxytryptamine in vibrated but not control arteries and abolished the vibration-induced depression in constrictor responses. The results suggest that acute vibration causes vascular dysfunction in digital arteries by increasing ROS levels, which is probably mediated by uncoupling of endothelial NOS. Therefore, therapeutic strategies to inhibit ROS or augment NO activity may be beneficial in HAVS.

Cold increases cutaneous vasoconstriction by unmasking the contractile activity of alpha(2C)-adrenoceptors (alpha(2C)-ARs) in vascular smooth muscle cells (VSMCs), which is mediated by the cold-induced mobilization of alpha(2C)-ARs from the transGolgi to the cell surface. The expression of alpha(2C)-ARs in human cutaneous VSMCs is under dual regulation by cyclic AMP: gene transcription is inhibited by cyclic AMP acting through protein kinase A but is increased by cyclic AMP acting through the exchange protein directly activated by cyclic AMP (EPAC) and the GTP-binding protein Rap1. Experiments were performed to further characterize the Rap1 signaling pathway. Forskolin (10 muM), the selective EPAC activator, 8-pCPT-2'-O-Me-cyclic AMP (CMC; 100 microM), or a constitutively active mutant of Rap1 (Rap1CA) increased the activity of c-Jun NH(2)-terminal kinase (JNK) in human cutaneous VSMCs. This was associated with the increased phosphorylation of c-Jun and activation of an activator protein (AP)-1 reporter construct, which were inhibited by the JNK inhibitor SP600125 (3 microM). Rap1CA increased the activity of an alpha(2C)-AR promoter-reporter construct, which was inhibited by SP600125 (3 microM) or by the mutation of an AP-1 binding site in the alpha(2C)-AR promoter. Furthermore, forskolin (10 microM) or CMC (100 microM) increased the expression of the alpha(2C)-AR protein, and these effects were inhibited by SP600125 (3 microM). Therefore, cyclic AMP increases the expression of alpha(2C)-ARs in cutaneous VSMCs by activating a novel Rap1 signaling pathway, mediated by the activation of JNK, AP-1, and the subsequent transcriptional activation of the alpha(2C)-AR gene. By increasing the expression of cold-responsive alpha(2C)-ARs, this pathway may contribute to enhanced cold-induced vasoconstriction in the cutaneous circulation, including Raynaud's phenomenon.

Latency Associated Peptide (LAP) binds TGF-beta1, forming a latent complex. Currently, LAP is presumed to function only as a sequestering agent for active TGF-beta1. Previous work shows that LAP can induce epithelial cell migration, but effects on leukocytes have not been reported. Because of the multiplicity of immunologic processes in which TGF-beta1 plays a role, we hypothesized that LAP could function independently to modulate immune responses. In separate experiments we found that LAP promoted chemotaxis of human monocytes and blocked inflammation in vivo in a murine model of the delayed-type hypersensitivity response (DTHR). These effects did not involve TGF-beta1 activity. Further studies revealed that disruption of specific LAP-thrombospondin-1 (TSP-1) interactions prevented LAP-induced responses. The effect of LAP on DTH inhibition depended on IL-10. These data support a novel role for LAP in regulating monocyte trafficking and immune modulation.

Because of the role of the RhoA/Rho kinase (ROCK) pathway in regulating numerous pathologic processes including vasoconstriction, vascular remodeling, and fibrosis, ROCK inhibitors may be especially beneficial in treating Raynaud's phenomenon and scleroderma.

Raynaud's phenomenon, which is characterized by intense cold-induced constriction of cutaneous arteries, is more common in women compared with men. Cold-induced constriction is mediated in part by enhanced activity of alpha(2C)-adrenoceptors (alpha(2C)-ARs) located on vascular smooth muscle cells (VSMs). Experiments were therefore performed to determine whether 17beta-estradiol regulates alpha(2C)-AR expression and function in cutaneous VSMs. 17beta-Estradiol (0.01-10 nmol/l) increased expression of the alpha(2C)-AR protein and the activity of the alpha(2C)-AR gene promoter in human cultured dermal VSMs, which was assessed following transient transfection of the cells with a promoter-reporter construct. The effect of 17beta-estradiol was associated with increased accumulation of cAMP and activation of the cAMP-responsive Rap2 GTP-binding protein. Transient transfection of VSMs with a dominant-negative mutant of Rap2 inhibited the 17beta-estradiol-induced activation of the alpha(2C)-AR gene promoter, whereas a constitutively active mutant of Rap2 increased alpha(2C)-AR promoter activity. The effects of 17beta-estradiol were inhibited by the estrogen receptor (ER) antagonist, ICI-182780 (1 micromol/l), and were mimicked by a cell-impermeable form of the hormone (estrogen:BSA) or by the selective ER-alpha receptor agonist 4,4',4'''-(4-propyl-[(1)H]-pyrazole-1,3,5-triyl)tris-phenol (PPT; 10 nmol/l) or the selective ER-beta receptor agonist 2,3-bis(4-hydroxyphenyl)-propionitrile (DPN; 10 nmol/l). Therefore, 17beta-estradiol increased expression of alpha(2C)-ARs by interacting with cell surface receptors to cause a cAMP/Rap2-dependent increase in alpha(2C)-AR transcription. In mouse tail arteries, 17beta-estradiol (10 nmol/l) increased alpha(2C)-AR expression and selectively increased the cold-induced amplification of alpha(2)-AR constriction, which is mediated by alpha(2C)-ARs. An estrogen-dependent increase in expression of cold-sensitive alpha(2C)-ARs may contribute to the increased activity of cold-induced vasoconstriction under estrogen-replete conditions.

Cutaneous vasoconstriction (VC), a critical thermoregulatory response to cold, is generally impaired with aging. However, the effects of aging on local cooling-induced VC and its underlying mechanisms are poorly understood. We tested whether aged skin exhibits attenuated localized cold-induced VC and whether Rho kinase-mediated cold-induced VC is augmented with age. Skin blood flow was monitored with laser Doppler flowmetry (LDF) on seven young and seven older subjects. Cutaneous vascular conductance (CVC; LDF/mean arterial pressure) was expressed as percentage change from baseline (%DeltaCVC(base)). In protocol 1, two forearm skin sites were cooled to six temperatures (31.5-19 degrees C) for 10 min each or two temperatures (29 degrees C, 24 degrees C) for 30 min each, with no age differences in the magnitude of VC. In protocol 2, three forearm skin sites were instrumented for intradermal microdialysis and cooled to 24 degrees C for 40 min. During minutes 1-5, there was no age difference in CVC responses at control sites (young: -45 +/- 6% vs. older: -46 +/- 3%, P > 0.9). Adrenoceptor antagonism (yohimbine + propranolol) abolished VC in young (to +15 +/- 13%, P < 0.05) but only partially inhibited VC in older subjects (to -23 +/- 6%, P < 0.05). Rho kinase inhibition plus adrenoceptor antagonism (yohimbine + propranolol + fasudil) abolished VC in both groups. During minutes 35-40, there was no age difference in control (young: -77 +/- 4% vs. older: -70 +/- 2%, P > 0.3) or adrenoceptor-antagonized responses (young: -61 +/- 3% vs. older: -55 +/- 2%, P > 0.3); however, Rho kinase inhibition plus adrenoceptor antagonism blocked more VC in older compared with young subjects (-19 +/- 11% vs. -35 +/- 3%, P < 0.05). Although its magnitude remains unaffected, cold-induced VC becomes less dependent on adrenergic and more dependent on Rho kinase signaling with advancing age.

Cutaneous vasoconstriction (VC) is the initial thermoregulatory response to cold exposure and can be elicited through either whole body or localized skin cooling. However, the mechanisms governing local cold-induced VC are not well understood. We tested the hypothesis that Rho kinase participates in local cold-induced cutaneous VC. In seven men and women (20-27 yr of age), up to four ventral forearm skin sites were instrumented with intradermal microdialysis fibers for localized drug delivery during cooling. Skin blood flow was monitored at each site with laser-Doppler flowmetry while local skin temperature was decreased and maintained at 24 degrees C for 40 min. Cutaneous vascular conductance (CVC; laser-Doppler flowmetry/mean arterial pressure) was expressed as percent change from 34 degrees C baseline. During the first 5 min of cooling, CVC decreased at control sites (lactated Ringer solution) to -45 +/- 6% (P < 0.001), increased at adrenoceptor-antagonized sites (yohimbine + propranolol) to 15 +/- 14% (P = 0.002), and remained unchanged at both Rho kinase-inhibited (fasudil) and adrenoceptor-antagonized + Rho kinase-inhibited sites (yohimbine + propranolol + fasudil) (-9 +/- 1%, P = 0.4 and -6 +/- 2%, P = 0.4, respectively). During the last 5 min of cooling, CVC further decreased at all sites when compared with baseline values (control, -77 +/- 4%, P < 0.001; adrenoceptor antagonized, -61 +/- 3%, P < 0.001; Rho kinase inhibited, -34 +/- 7%, P < 0.001; and adrenoceptor antagonized + Rho kinase inhibited sites, -35 +/- 3%, P < 0.001). Rho kinase-inhibited and combined treatment sites were significantly attenuated when compared with both adrenoceptor-antagonized (P < 0.01) and control sites (P < 0.0001). Rho kinase mediates both early- and late-phase cold-induced VC, supporting in vitro findings and providing a putative mechanism through which both adrenergic and nonadrenergic cold-induced VC occurs in an in vivo human thermoregulatory model.

Experiments were performed to determine the effects of endothelial denudation in vivo on vasoconstrictor responses of mouse tail artery segments in vitro. A sterile wire (70 microm diameter) was inserted into tail arteries of anesthetized mice to mechanically denude the endothelium, and the animals were allowed to recover for 48 hours. The function of pressurized tail artery segments was then studied in vitro. Intimal injury markedly reduced endothelium-dependent relaxation to acetylcholine. Constriction evoked by the selective alpha1-adrenoceptor (alpha1-AR) agonist, phenylephrine, was not affected by in vivo endothelial denudation, indicating that the contractile function of vascular smooth muscle cells (VSMCs) was not impaired. However, constriction to the selective alpha2-AR agonist UK14304 or to endothelin-1 was significantly inhibited. Confocal microscopy of intact tail arteries localized caveolin-1 to punctuate structures, arranged in rows on or close to the surface of VSMCs. After in vivo endothelial denudation, this pattern was disrupted and caveolin-1 was localized to intracellular sites. When VSMC caveolae were disrupted in control arteries using the cholesterol acceptor methyl-beta-cyclodextrin, there was a similar impairment in constriction to endothelin-1 or alpha2-AR stimulation, but not alpha1-AR activation. These results suggest that intimal injury to small cutaneous arteries disrupts VSMC surface caveolae and selectively impairs constriction to stimuli that are dependent on these structures for signaling.

The imbalance theory proposes that arterial thrombosis is dependent on a ratio or balance between the cyclooxygenase (COX)-1-dependent generation of thromboxane in platelets and the COX-2-dependent generation of prostacyclin in the endothelium. Accordingly, by reducing endothelium-derived prostacyclin levels, selective COX-2 inhibitors would increase susceptibility to vasoconstriction, platelet activation and atherothrombosis. This imbalance theory has been frequently used to legitimize the potential for increased cardiovascular risk associated with the use of these agents. Surprisingly, the imbalance theory is based not on human vascular biology but on the analysis of urinary prostacyclin metabolites. In this review, I discuss the theory in the context of direct studies of human vascular biology, which indicate that the imbalance theory is based on incorrect assumptions and is not plausible.

Reactive oxygen species, including superoxide, are important mediators of the pathophysiology of hypertension. In the vasculature, superoxide antagonizes nitric oxide (NO*), resulting in increased vascular tone. The GTP binding protein Rac regulates a wide variety of cellular functions, including the activation of NADPH oxidase, the major source of O2*-in the blood vessel wall. An hypothesis is that Rac1 may act as an important regulator of vascular O2*- production, contributing to the balance between O2*- and NO* and maintaining consequent homeostasis of blood pressure. To alter the activity of vascular NADPH oxidase, the authors developed a transgenic animal model that overexpresses the human cDNA of the constitutively active mutant of Rac1 (RacCA) in smooth muscle cells using the smooth muscle +/--actin promoter. The RacCA transgenic had excessive amounts of O2*- in the vessel wall that, which led to heightened production of peroxynitrite, as detected by increased protein nitration and reduced NO* levels. RacCA mice developed moderate hypertension, which was corrected by N-acetyl-L-cysteine (NAC). RacCA transgenic mice also developed left ventricular hypertrophy as a secondary effect of pressure overload. The data suggest that Rac1 is a critical regulator of the redox state of blood vessels and homeostasis of blood pressure.

Recent studies using ovarian cancer cells have shown that the catecholamine hormones norepinephrine (norepi) and epinephrine (epi) may influence cancer progression by modulating the expression of matrix metalloproteinases (MMP) and vascular endothelial growth factor (VEGF). The purpose of this study is to determine if the stress hormone norepi can influence the expression of MMP-2, MMP-9, and VEGF in nasopharyngeal carcinoma (NPC) tumors by using three NPC tumor cell lines. The NPC cell lines HONE-1, HNE-1, and CNE-1 were treated with norepi. The effects of norepi on MMP-2, MMP-9, and VEGF synthesis were measured by ELISA; functional MMP activity was measured by the invasive potential of the cells using a membrane invasion culture system whereas functional activity of VEGF was analyzed using a human umbilical vein endothelial cell tube formation assay. Norepi treatment increased MMP-2, MMP-9, and VEGF levels in culture supernatants of HONE-1 cells, which could be inhibited by the beta-blocker propranolol. Norepi induced the invasiveness of all NPC cell lines in a dose-dependent manner, which was blocked by CMT-3, an MMP inhibitor, and propranolol. Norepi stimulated the release of functional angiogenic VEGF by HONE-1 cells as well. Finally, HONE-1 cells were shown to express beta-adrenergic receptors as did seven of seven NPC biopsies examined. The data suggest that catecholamine hormones produced by the sympathetic-adrenal medullary axis may affect NPC tumor progression, in part, through modulation of key angiogenic cytokines.

The aim of this study was to determine whether prolonged loss of NO activity, in endothelial NO synthase knockout (eNOS(-/-)) mice, influences endothelin (ET) ETA receptor-mediated smooth muscle contraction and, if so, to define the underlying mechanism(s). In isolated endothelium-denuded abdominal aortas, contractions to the selective ETA receptor agonist ET-1(1-31) were significantly increased in aortas from eNOS(-/-) compared with wild-type (WT) mice. In contrast, contractions to the alpha1-adrenergic agonist phenylephrine or the thromboxane (TX) A2 analog U-46619 were similar between eNOS(-/-) and WT mice. Immunofluorescent and Western blot analysis demonstrated that the aortic expression of ETA receptors was decreased in eNOS(-/-) compared with WT mice. Contractions evoked by ET-1(1-31), but not phenylephrine, were reduced by inhibition of cyclooxygenase-2 (COX-2) (indomethacin or celecoxib) or of TXA2/prostaglandin H2 receptors (SQ-29548). After COX inhibition, contractions to ET-1(1-31) were no longer increased and were actually decreased in eNOS(-/-) compared with WT aortas. Western blot analysis revealed that endothelium-denuded abdominal aortas express COX-2, but not COX-1, and that expression of COX-2 was significantly increased in eNOS(-/-) compared with WT mice. Contractions to the COX substrate arachidonic acid were also increased in eNOS(-/-) aortas. Furthermore, ET-1(1-31) but not phenylephrine stimulated production of the TXA2 metabolite TXB2, which was increased in eNOS(-/-) compared with WT aortas. Therefore, COX-2 plays a crucial and selective role in ETA-mediated smooth muscle contraction. Furthermore, COX-2 expression is increased in eNOS(-/-) mice, which overcomes a reduced expression of ETA receptors and enables a selective increase in contraction to ETA receptor stimulation.

The vascular symptoms of hand-arm vibration syndrome, including cold-induced vasospasm, are in part mediated by increased sensitivity of cutaneous arteries to sympathetic stimulation. The goal of the present study was to use a rat tail model to analyze the effects of vibration on vascular function and alpha-adrenoceptor (AR) responsiveness. Rats were exposed to a single period of vibration (4 h, 125 Hz, constant acceleration 49 m/s2 root mean square). The physical or biodynamic response of the tail demonstrated increased transmissibility or resonance at this frequency, similar to that observed during vibration of human fingers. Morphological analysis demonstrated that vibration did not appear to cause structural injury to vascular cells. In vitro analysis of vascular function demonstrated that constriction to the alpha1-AR agonist phenylephrine was similar in vibrated and control arteries. In contrast, constriction to the alpha2-AR agonist UK14304 was increased in vibrated compared with control arteries, both in endothelium-containing or endothelium-denuded arteries. The alpha2C-AR antagonist MK912 (3 x 10(-10) M) inhibited constriction to UK14304 in vibrated but not control arteries, reversing the vibration-induced increase in alpha2-AR activity. Moderate cooling (to 28 degrees C) increased constriction to the alpha2-AR agonist in control and vibrated arteries, but the magnitude of the amplification was less in vibrated compared with control arteries. Endothelium-dependent relaxation to acetylcholine was similar in control and vibrated arteries. Based on these results, we conclude that a single exposure to vibration caused a persistent increase in alpha2C-AR-mediated vasoconstriction, which may contribute to the pathogenesis of vibration-induced vascular disease.

The present study evaluated mechanical stretch-induced apoptosis in swine vascular smooth muscle cells (VSMC) of different phenotypes. We demonstrated that differentiated VSMC express a greater level of Bcl-2-associated death factor (BAD) and have a significant cell loss when exposed to mechanical stretch (10% elongation, 1 Hz) for 24 h. We further demonstrated that apoptosis was significantly increased only in differentiated VSMC exposed to mechanical stretch. To test the hypothesis that the intracellular level of BAD in VSMC determines its response to mechanical stretch-induced apoptosis, we examined whether BAD expression was upregulated by mechanical stretch-induced apoptosis and was associated with the increase in the apoptosis level of differentiated VSMC. When exposed to mechanical stretch, the expression of BAD in differentiated VSMC was elevated at 1 h and remained at higher levels during the application of stretch (24 h). In contrast, Bcl-2 expression was suppressed during the application of stretch. Moreover, the proapoptotic function of BAD was inhibited by overexpression of Bcl-2 through transient transfection of VSMC with pCEP4-Bcl-2 or incubation of VSMC with vascular epithelial growth factor. These results suggest that mechanical stretch-induced VSMC apoptosis is phenotype dependent. The higher levels of apoptosis of differentiated VSMC upon mechanical stretch were, at least in part, dependent on their intrinsic level of BAD., (Copyright 2006 S. Karger AG, Basel.)

The goal of this study was to determine whether acetylcholine evokes endothelium-dependent contraction in mouse arteries and to define the mechanisms involved in regulating this response. Arterial rings isolated from wild-type (WT) and endothelial nitric oxide (NO) synthase knockout (eNOS(-/-)) mice were suspended for isometric tension recording. In abdominal aorta from WT mice contracted with phenylephrine, acetylcholine caused a relaxation that reversed at the concentration of 0.3-3 microM. After inhibition of NO synthase [with N(omega)-nitro-l-arginine methyl ester (l-NAME), 1 mM], acetylcholine (0.1-10 microM) caused contraction under basal conditions or during constriction to phenylephrine, which was abolished by endothelial denudation. This contraction was inhibited by the cyclooxygenase inhibitor indomethacin (1 muM) or by a thromboxane A(2) (TxA(2)) and/or prostaglandin H(2) receptor antagonist SQ-29548 (1 microM) and was associated with endothelium-dependent generation of the TxA(2) metabolite TxB(2.) Also, SQ-29548 (1 microM) abolished the reversal in relaxation evoked by 0.3-3 microM acetylcholine and subsequently enhanced the relaxation to the agonist. The magnitude of the endothelium-dependent contraction to acetylcholine (0.1-10 microM) was similar in aortas from WT mice treated in vitro with l-NAME and from eNOS(-/-) mice. In addition, we found that acetylcholine (10 microM) also caused endothelium-dependent contraction in carotid and femoral arteries of eNOS(-/-) mice. These results suggest that acetylcholine initiates two competing responses in mouse arteries: endothelium-dependent relaxation mediated predominantly by NO and endothelium-dependent contraction mediated most likely by TxA(2).

Cold constricts cutaneous blood vessels by selectively increasing the activity of smooth muscle alpha2-adrenoceptors (alpha2-ARs). In mouse tail arteries, alpha2-AR constriction is mediated by alpha2A-ARs at 37 degrees C, whereas the cold-induced augmentation in alpha2-AR activity is mediated entirely by alpha2C-ARs. Cold causes translocation of alpha2C-ARs from the trans-Golgi to the plasma membrane, mediated by cold-induced activation of RhoA and Rho kinase. The present experiments analyzed the mechanisms underlying these responses. Mouse tail arteries were studied in a pressure myograph. Cooling the arteries (28 degrees C) caused a rapid increase in reactive oxygen species (ROS) in smooth muscle cells, determined by confocal microscopy of arteries loaded with the ROS-sensitive probes, dichlorodihydrofluorescein or reduced Mitotracker Red. The inhibitor of mitochondrial complex I rotenone (10 micromol/l), the antioxidant N-acetylcysteine (NAC; 20 mmol/l), or the cell-permeable mimic of superoxide dismutase MnTMPyP (50 micromol/l) did not affect vasoconstriction to alpha2-AR stimulation (UK-14304) at 37 degrees C but dramatically inhibited the response at 28 degrees C. Indeed, these ROS inhibitors abolished the cold-induced increase in alpha2-AR constrictor activity. NAC (20 mmol/l) or MnTMPyP (50 micromol/l) also abolished the cold-induced activation of RhoA in human cultured vascular smooth muscle cells and the cold-induced mobilization of alpha2C-ARs to the cell surface in human embryonic kidney 293 cells transfected with the receptor. The combined results suggest that cold-induced constriction is mediated by redox signaling in smooth muscle cells, initiated by mitochondrial generation of ROS, which stimulate RhoA/Rho kinase signaling and the subsequent mobilization of alpha2C-ARs to the cell surface. Altered activity of ROS may contribute to cold-induced vasospasm occurring in Raynaud's phenomenon.

Phenylpropanolamine (dl-norephedrine) was one of the most widely used therapeutic agents to act on the sympathetic nervous system. Because of concerns regarding incidents of stroke, its use as a nasal decongestant was discontinued. Although considered an alpha1-adrenergic agonist, the vascular adrenergic pharmacology of phenylpropanolamine was not fully characterized. Unlike most other circulations, the vasculature of the nasal mucosa is highly enriched with constrictor alpha2-adrenoceptors. Therefore, experiments were performed to determine whether phenylpropanolamine activates vascular alpha2-adrenoceptors. Mouse tail and mesenteric small arteries and human small dermal veins were isolated and analyzed in a perfusion myograph. The selective alpha1-adrenergic agonist phenylephrine caused constriction of tail and mesenteric arteries and human veins. The selective alpha2-adrenergic agonist UK14,304 [5-bromo-N-(4,5-dihydro-1H-imidazol-2-yl)-6-quinoxalinamine] caused constriction in tail arteries and in human veins, but not mesenteric arteries. The lack of constriction to UK14,304 was also observed in endothelium-denuded mesenteric arteries. Phenylpropanolamine constricted both types of artery but was 62-fold more potent in tail arteries. In mesenteric arteries, constriction to phenylpropanolamine was not affected by the selective alpha2-adrenergic antagonist, rauwolscine (10(-7) M) but was abolished by the selective alpha1-adrenergic antagonist, prazosin (3 x 10(-7) M). In contrast, constriction to phenylpropanolamine in tail arteries and in human veins was inhibited by rauwolscine but not prazosin. Therefore, phenylpropanolamine is a preferential alpha2-adrenergic agonist. At low concentrations, it constricts blood vessels that express functional alpha2-adrenoceptors, whereas at much higher concentrations, phenylpropanolamine also activates vascular alpha1-adrenoceptors. This action likely contributed to phenylpropanolamine's therapeutic activity, namely constriction of the nasal vasculature.

Experiments were performed to determine whether remodeling of the actin cytoskeleton contributes to arteriolar constriction. Mouse tail arterioles were mounted on cannulae in a myograph and superfused with buffer solution. The alpha1-adrenergic agonist phenylephrine (0.1-1 micromol/l) caused constriction that was unaffected by cytochalasin D (300 nmol/l) or latrunculin A (100 nmol/l), inhibitors of actin polymerization. In contrast, each compound abolished the mechanosensitive constriction (myogenic response) evoked by elevation in transmural pressure (PTM; 10-60 or 90 mmHg). Arterioles were fixed, permeabilized, and stained with Alexa-568 phalloidin and Alexa-488 DNAse I to visualize F-actin and G-actin, respectively, using a Zeiss 510 laser scanning microscope. Elevation in PTM, but not phenylephrine (1 micromol/l), significantly increased the intensity of F-actin and significantly decreased the intensity of G-actin staining in arteriolar vascular smooth muscle cells (VSMCs). The increase in F-actin staining caused by an elevation in PTM was inhibited by cytochalasin D. In VSMCs at 10 mmHg, prominent F-actin staining was restricted to the cell periphery, whereas after elevation in PTM, transcytoplasmic F-actin fibers were localized through the cell interior, running parallel to the long axis of the cells. Phenylephrine (1 micromol/l) did not alter the architecture of the actin cytoskeleton. In contrast to VSMCs, the actin cytoskeleton of endothelial or adventitial cells was not altered by an elevation in PTM. Therefore, the actin cytoskeleton of VSMCs undergoes dramatic alteration after elevation in PTM of arterioles and plays a selective and essential role in mechanosensitive myogenic constriction.