Mouse Mesenteric Resistance Arteries Research Articles

Myoendothelial feedback in mouse mesenteric resistance arteries is similar between the sexes, dependent on nitric oxide synthase, and independent of TPRV4.

Myoendothelial feedback (MEF), the endothelium-dependent vasodilation following sympathetic vasoconstriction (mediated by smooth muscle to endothelium gap junction communication), has been well studied in resistance arteries of males, but not females. We hypothesized that MEF responses would be similar between the sexes, but different in the relative contribution of the underlying nitric oxide and hyperpolarization mechanisms, given that these mechanisms differ between the sexes in agonist-induced endothelium-dependent dilation. We measured MEF responses (diameter changes) of male and female first- to second-order mouse mesenteric arteries to phenylephrine (10 µM) over 30 min using isolated pressure myography ± blinded inhibition of nitric oxide synthase (NOS) using Nω-nitro-l-arginine methyl ester (l-NAME; 0.1-1.0 mM), hyperpolarization using 35 mM KCl, or transient receptor potential vanilloid 4 (TRPV4) channels using GSK219 (0.1-1.0 µM) or RN-1734 (30 µM). MEF was similar [%dilation (means ± SE): males = 26.7 ± 2.0 and females = 26.1 ± 1.9 at 15 min] and significantly inhibited by l-NAME (1.0 mM) at 15 min [%dilation (means ± SE): males = 8.2 ± 3.3, P < 0.01; females = 6.8 ± 1.9, P < 0.001] and over time (P < 0.01) in both sexes. l-NAME (0.1 mM) + 35 mM KCl nearly eliminated MEF in both sexes (P < 0.001-0.0001). Activation of TRPV4 with GSK101 (0.1-10 µM) induced similar dilation between the sexes. Inhibition of TRPV4, which is reportedly involved in the hyperpolarization mechanism, did not inhibit MEF in either sex. Similar expression of eNOS was found between the sexes with Western blot. Thus, MEF is prominent and similar in murine first- and second-order mesenteric resistance arteries of both sexes, and reliant primarily on NOS and secondarily on hyperpolarization, but not TRPV4.NEW & NOTEWORTHY We found that female mesenteric resistance arteries have similar postconstriction dilatory responses (i.e., myoendothelial feedback) to a sympathetic neurotransmitter analog as male arteries. Both sexes use nitric oxide synthase (NOS) and hyperpolarization, but not TRPV4, in this response. Moreover, the key protein involved in this pathway (eNOS) is similarly expressed in these arteries between the sexes. These similarities are surprising given that agonist-induced endothelium-dependent dilatory mechanisms differ in these arteries between the sexes.

FRESH 3D Bioprinted Collagen‐based Resistance Vessels and Multiscale Vascular Microfluidics

The pursuit of an ideal model system to study the pathophysiology of vascular disease has included the use of tissue engineered blood vessels (TEBV), vascularizing engineered volumetric tissue, silicone‐based “organ‐on‐a‐chip” platform, and tissue decellularization; yet we are still far from generating engineered vascular tissue that recapitulate native physiology. Despite advances such as 3D bioprinting that have led to new approaches for creating fluidic channels, it remains challenging to produce continuous networks with vessels ranging from large (&gt;6 mm) to small (&lt;1 mm) using native‐like extracellular matrix (ECM) that recapitulates the mechanical properties, geometric organization, and complexity of the cellular and extracellular microenvironment. An ideal engineering strategy would combine the controlled fluid flow achieved by organ‐on‐a‐chip platforms with the ECM structure, composition, and biomechanics of decellularized tissue. Here, using Freeform Reversible Embedding of Suspended Hydrogels (FRESH) 3D bioprinting, we spatially define ECM composition and vascular geometry to create collagen‐based microfluidics and an engineered resistance artery scaffold to study how ECM composition, mechanical properties, and fluid flow contribute to vascular disease. A 3D vascular microfluidic CAD model was designed with a hierarchically branching network containing a single inlet and outlet of 1 mm that branched down to 250 µm channels embedded within a collagen scaffold. This model was FRESH printed with 24 mg/mL collagen‐I and yielded a high‐fidelity scaffold with patent internal vasculature. 3D imaging and gauging analysis of the CAD model compared to the collagen scaffold revealed an average positive deviation of 9.9 µm and an average negative deviation of 56.3 µm. To interface with and perfuse the soft collagen microfluidic scaffold we 3D printed a custom bioreactor chamber for long‐term culture capable of &gt;1‐week perfusion under physiological conditions. In addition to embedded vascular networks, FRESH bioprinting allows for direct fabrication of suspended vessels. We designed an ECM scaffold scaled to match a resistance vessel consisting of a collagen‐I frame and suspended vessel (inner diameter of 300 µm, outer diameter of 500 µm). The collagen frame provides overall scaffold stability and allows interfacing to the custom perfusion bioreactor. Using multimaterial FRESH printing, we varied the ECM composition by patterning fibronectin along the inner lumen and collagen‐I within the outer adventitial wall. Biomechanical evaluation using pressure and wire myography revealed a burst pressure &gt;140 mmHg and an average peak tension of 600 mg, comparable to mouse mesenteric resistance arteries. Together, this work highlights the exciting potential for ECM‐based microfluidic scaffolds and vascular tissue engineering that will set the foundation for future work to replicate a healthy and hypertensive vessel microenvironment. In addition to vascular biology, the collagen‐based microfluidics are amenable to other organ systems, and can be utilized to study cell proliferation, metastasis, and ECM remodeling in a 3D environment.

A17398 Nitric oxide synthase regulates of the anti-contractile functions of perivascular adipose tissue

Objectives: Perivascular adipose tissue (PVAT) plays a crucial role in the modulation of vascular tone. Loss of function in obesity leads to an increase in arterial tone, and may contribute to development of hypertension. Activation of nitric oxide synthase (NOS) has been implicated in the anti-contractile effect of PVAT. Accordingly, we have examined the roles of NOS in healthy and obese PVAT. Methods: Electrical field stimulation profiles or noradrenaline concentration response curves were generated in healthy, obese, and eNOS−/− mouse mesenteric resistance arteries. Pharmacological manipulation of NOS was conducted using the non-specific inhibitor; L-NMMA, and activator; histamine. The role of β3-adrenoceptors was investigated using the agonist CL-316,243. Immunohistochemistry was use to examine expression of the endothelial NOS isoform (eNOS) in PVAT. Results: Stimulation of PVAT induced an anti-contractile effect on healthy mouse arteries, which was absent in obese and eNOS−/− mouse arteries. Inhibition of NOS in healthy mouse PVAT abolished the anti-contractile effect, and activation of NOS in obese PVAT restored the anti-contractile effect. Using immunohistochemistry, eNOS is the isoform present in mesenteric PVAT, and expression is unchanged in obesity. The β3-adrenoceptor agonist enhanced the anti-contractile effect, and this enhancement was abolished using the NOS inhibitor. Conclusion: These results demonstrate that the β3-adrenoceptor mediated anti-contractile effect of PVAT is dependent upon NOS. In obesity, the anti-contractile effect is lost which may contribute to development of hypertension. However, function can be rescued using activation of NOS; presenting a potential pharmacological target for intervention in obesity-related hypertension.

Activation of the β-common receptor by erythropoietin impairs acetylcholine-mediated vasodilation in mouse mesenteric arterioles.

Clinically, erythropoietin (EPO) is known to increase systemic vascular resistance and arterial blood pressure. However, EPO stimulates the production of the potent vasodilator, nitric oxide (NO), in culture endothelial cells. The mechanism by which EPO causes vasoconstriction despite stimulating NO production may be dependent on its ability to activate two receptor complexes, the homodimeric EPO (EPOR 2) and the heterodimeric EPOR/β‐common receptor (β CR). The purpose of this study was to investigate the contribution of each receptor to the vasoactive properties of EPO. First‐order, mesenteric arteries were isolated from 16‐week‐old male C57BL/6 mice, and arterial function was studied in pressure arteriographs. To determine the contribution of each receptor complex, EPO‐stimulating peptide (ESP), which binds and activates the heterodimeric EPOR/β CR complex, and EPO, which activates both receptors, were added to the arteriograph chamber 20 min prior to evaluation of endothelium‐dependent (acetylcholine, bradykinin, A23187) and endothelium‐independent (sodium nitroprusside) vasodilator responses. Only ACh‐induced vasodilation was impaired in arteries pretreated with EPO or ESP. EPO and ESP pretreatment abolished ACh‐induced vasodilation by 100% and 60%, respectively. EPO and ESP did not affect endothelium‐independent vasodilation by SNP. Additionally, a novel β CR inhibitory peptide (β IP), which was computationally developed, prevented the impairment of acetylcholine‐induced vasodilation by EPO and ESP, further implicating the EPOR/β CR complex. Last, pretreatment with either EPO or ESP did not affect vasoconstriction by phenylephrine and KCl. Taken together, these findings suggest that acute activation of the heterodimeric EPOR/β CR in endothelial cells leads to a selective impairment of ACh‐mediated vasodilator response in mouse mesenteric resistance arteries.

Role of Sympathetic Nerves and Adipocyte Catecholamine Uptake in the Vasorelaxant Function of Perivascular Adipose Tissue.

Healthy perivascular adipose tissue (PVAT) exerts an anticontractile effect on resistance arteries which is vital in regulating arterial tone. Activation of β3-adrenoceptors by sympathetic nerve-derived NA (noradrenaline) may be implicated in this effect and may stimulate the release of the vasodilator adiponectin from adipocytes. Understanding the mechanisms responsible is vital for determining how PVAT may modify vascular resistance in vivo. Electrical field stimulation profiles of healthy C57BL/6J mouse mesenteric resistance arteries were characterized using wire myography. During electrical field stimulation, PVAT elicits a reproducible anticontractile effect, which is endothelium independent. To demonstrate the release of an anticontractile factor, the solution surrounding stimulated exogenous PVAT was transferred to a PVAT-denuded vessel. Post-transfer contractility was significantly reduced confirming that stimulated PVAT releases a transferable anticontractile factor. Sympathetic denervation of PVAT using tetrodotoxin or 6-hydroxydopamine completely abolished the anticontractile effect. β3-adrenoceptor antagonist SR59203A reduced the anticontractile effect, although the PVAT remained overall anticontractile. When the antagonist was used in combination with an OCT3 (organic cation transporter 3) inhibitor, corticosterone, the anticontractile effect was completely abolished. Application of an adiponectin receptor-1 blocking peptide significantly reduced the anticontractile effect in +PVAT arteries. When used in combination with the β3-adrenoceptor antagonist, there was no further reduction. In adiponectin knockout mice, the anticontractile effect is absent. The roles of PVAT are 2-fold. First, sympathetic stimulation in PVAT triggers the release of adiponectin via β3-adrenoceptor activation. Second, PVAT acts as a reservoir for NA, preventing it from reaching the vessel and causing contraction.

Abstract P500: Activation of the Heterodimeric Erythropoietin /β-Common Receptor Impairs Acetylcholine Mediated Vasodilation in Mouse Mesenteric Arterioles

Erythropoietin (EPO) increases systemic vascular resistance and blood pressure. However, endothelial cells cultured in the presence of EPO demonstrate increased production of the potent vasodilator, nitric oxide (NO). The mechanism by which EPO causes vasoconstriction despite stimulating NO production may be dependent on its ability to differentially activate the two receptor complexes, the homodimeric EPO (EPOR 2 ) and the heterodimeric EPOR/β-common receptor (βCR). Objective: The purpose of this study was to investigate the contribution of the EPOR 2 and βCR receptor to the vasoactive properties of EPO. Methods: First order, mesenteric arteries isolated from 16-week old male C57BL/6 mice were cannulated and perfused using a pressure arteriography system. To determine the contribution of each receptor complex, arteries were incubated with EPO stimulating peptide (ESP) which binds and activates only the heterodimeric EPOR/βCR complex or EPO which activates both receptors, 20 min prior to evaluation of vasoconstrictor (phenylephrine and potassium chloride), endothelium-dependent (acetylcholine, bradykinin, A23187) and -independent (sodium nitroprusside) vasodilator responses. Additionally, we studied the effect of a novel βCR inhibitory peptide (βIP) which was developed in silico and validated by demonstrating that it selectively inhibits binding of ligands to the βCR. Results: Acetylcholine induced vasodilation was impaired in arteries pretreated with EPO or ESP by 100% and 60%, respectively. EPO and ESP did not affect endothelium-dependent vasodilation by Bradykinin or A23187, endothelium-independent vasodilation by sodium nitroprusside, or vasoconstriction by phenylephrine and KCl. The βIP prevented the impairment of acetylcholine-induced vasodilation by EPO and ESP. Conclusion: Together, our findings suggest that activation of the heterodimeric EPOR/βCR leads to selective impairment of ACh-mediated vasodilator response in mouse mesenteric resistance arteries. Thus the βCR might have a role in mediating hypertensive effects of EPO. Therapeutic inhibition of the βCR might prevent vascular complications of EPO without affecting erythropoiesis.

Inwardly rectifying K+ channels are major contributors to flow-induced vasodilatation in resistance arteries.

Endothelial inwardly rectifying K+ (Kir2.1) channels regulate flow-induced vasodilatation via nitric oxide (NO) in mouse mesenteric resistance arteries. Deficiency of Kir2.1 channels results in elevated blood pressure and increased vascular resistance. Flow-induced vasodilatation in human resistance arteries is also regulated by inwardly rectifying K+ channels. This study presents the first direct evidence that Kir channels play a critical role in physiological endothelial responses to flow. Inwardly rectifying K+ (Kir) channels are known to be sensitive to flow, but their role in flow-induced endothelial responses is not known. The goal of this study is to establish the role of Kir channels in flow-induced vasodilatation and to provide first insights into the mechanisms responsible for Kir signalling in this process. First, we establish that primary endothelial cells isolated from murine mesenteric arteries express functional Kir2.1 channels sensitive to shear stress. Then, using the Kir2.1+/- heterozygous mouse model, we establish that downregulation of Kir2.1 results in significant decrease in shear-activated Kir currents and inhibition of endothelium-dependent flow-induced vasodilatation (FIV) assayed in pressurized mesenteric arteries pre-constricted with endothelin-1. Deficiency in Kir2.1 also results in the loss of flow-induced phosphorylation of eNOS and Akt, as well as inhibition of NO generation. All the effects are fully rescued by endothelial cell (EC)-specific overexpression of Kir2.1. A component of FIV that is Kir independent is abrogated by blocking Ca2+ -sensitive K+ channels. Kir2.1 has no effect on endothelium-independent and K+ -induced vasodilatation in denuded arteries. Kir2.1+/- mice also show increased mean blood pressure measured by carotid artery cannulation and increased microvascular resistance measured using a tail-cuff. Importantly, blocking Kir channels also inhibits flow-induced vasodilatation in human subcutaneous adipose microvessels. Endothelial Kir channels contribute to FIV of mouse mesenteric arteries via an NO-dependent mechanism, whereas Ca2+ -sensitive K+ channels mediate FIV via an NO-independent pathway. Kir2 channels also regulate vascular resistance and blood pressure. Finally, Kir channels also contribute to FIV in human subcutaneous microvessels.

Central Role of P2Y 6 UDP Receptor in Arteriolar Myogenic Tone

Objective— Myogenic tone (MT) of resistance arteries ensures autoregulation of blood flow in organs and relies on the intrinsic property of smooth muscle to contract in response to stretch. Nucleotides released by mechanical strain on cells are responsible for pleiotropic vascular effects, including vasoconstriction. Here, we evaluated the contribution of extracellular nucleotides to MT. Approach and Results— We measured MT and the associated pathway in mouse mesenteric resistance arteries using arteriography for small arteries and molecular biology. Of the P2 receptors in mouse mesenteric resistance arteries, mRNA expression of P2X 1 and P2Y 6 was dominant. P2Y 6 fully sustained UDP/UTP-induced contraction (abrogated in P2ry6 −/− arteries). Preventing nucleotide hydrolysis with the ectonucleotidase inhibitor ARL67156 enhanced pressure-induced MT by 20%, whereas P2Y 6 receptor blockade blunted MT in mouse mesenteric resistance arteries and human subcutaneous arteries. Despite normal hemodynamic parameters, P2ry6 −/− mice were protected against MT elevation in myocardial infarction–induced heart failure. Although both P2Y 6 and P2Y 2 receptors contributed to calcium mobilization, P2Y 6 activation was mandatory for RhoA–GTP binding, myosin light chain, P42–P44, and c-Jun N-terminal kinase phosphorylation in arterial smooth muscle cells. In accordance with the opening of a nucleotide conduit in pressurized arteries, MT was altered by hemichannel pharmacological inhibitors and impaired in Cx43 +/− and P2rx7 −/− mesenteric resistance arteries. Conclusions— Signaling through P2 nucleotide receptors contributes to MT. This mechanism encompasses the release of nucleotides coupled to specific autocrine/paracrine activation of the uracil nucleotide P2Y 6 receptor and may contribute to impaired tissue perfusion in cardiovascular diseases.

0362: Interleukin 17 production induced by angiotensin II type 2 receptor activation in T cells drives flow-mediated outward remodeling of mouse resistance arteries

Flow (shear stress)-mediated outward remodeling of resistance arteries is involved in collateral growth during postischemic revascularization. Besides the NO pathway the involvement of inflammatory factors and reactive oxygen species has been suggested in this remodeling although the mechanism and the sequence remains unknown. As T cells drive the inflammatory response, we investigated their role in flow-mediated remodeling. Mouse mesenteric resistance arteries (250μm internal diameter) were submitted in vivo to a chronic increase (144±18 to 239±25 μl/min) in blood flow. Arteries were collected for in vitro analysis after 1 to 7 days. After 1 week, remodeling occurred in high flow (HF) arteries (diameter increased from 242±21μm to 324±20μm, n=xx per group, P<00.1) in control mice. No remodeling occurred in nude mice and in mice treated with anti-CD3 antibodies. In control arteries lymphocyte T cells accumulate around HF arteries at day 1 (shown using both immunohistology and flow cytometry) and they expressed angiotensin II type 2 receptor (AT2R). In isolated T cells AT2R stimulation induced preferentially IL-17 production (20±6pg/ml to 50±5pg/ml, n=xx per group, P<00.1). We then tested the occurrence of remodeling in mice lacking AT2R or IL-17. In both AT2R-/- and IL-17-/- mice diameter expansion did not occur whereas T cells and myeloid cells accumulation around the HF artery was not affected. IL-17 infusion (5ng/h, Alzet osmotic minipump) to nude mice and to AT2R-/- mice restored HF remodeling to control level. In order to confirm the role of AT2R in flow-mediated remodeling in another vascular territory, neovascularization was examined using an ischemic skin flap model in AT2R-/- mice. As in mesenteric arteries, the absence of AT2R prevented diameter expansion in the arteries feeding the skin flap. we demonstrate for the first time the involvement of lymphocytes T cells polarization into TH17 by angiotensin II type 2 receptor in flow-mediated outward remodeling of resistance arteries.

Chronic intermittent hypoxia alters blood pressure and mesenteric small resistance arterial responsiveness in mice (710.9)

Blood pressure (BP) is elevated in rats and mice exposed to chronic intermittent hypoxia (CIH). It is unclear if altered vascular function, such as myogenic reactivity (MR), contributes to BP elevation in CIH. We aimed to characterize the vascular alterations in small resistance arteries from CIH‐exposed mice. C57BL/6J mice were exposed to either CIH (1 min alterations of 5% and 21% O2) or normoxia (sham control, constant 21% O2), 8 hrs/day for 5 weeks. Compared to sham controls, CIH‐exposed mice had significantly higher BP, measured by radio telemetry. Mesenteric small resistance arteries were isolated and pressurized for diameter and wall thickness measurement. Myogenic constriction in response to intraluminal pressure increase (i.e., MR) was significantly attenuated, especially at pressures 蠅 70 mm Hg. At 70 mm Hg, vasodilations to acetylcholine and sodium nitroprusside, but not to external Ca2+ removal, were significantly impaired in CIH, whereas vasoconstrictions to phenylephrine (PE), endothelin‐1, U46619, and high [K+]o were similar between sham and CIH arteries. When intraluminal pressure was increased to 110 mm Hg, however, PE‐induced vasoconstriction was significantly reduced, and nitric oxide‐dependent vasodilation was further attenuated in CIH arteries. Arterial remodeling was not obvious since wall thickness, wall to lumen ratio, and passive diameters in Ca2+‐free solution were similar between two groups. Thus, CIH 1) elevates BP; 2) reduces MR; 3) reduces arterial responsiveness to vasodilators, and, at higher intraluminal pressure, to vasoconstrictors; 4) has no effect on arterial remodeling in mouse mesenteric small resistance arteries. It is likely that the altered arterial responsiveness is involved in the regulation of BP in CIH.Grant Funding Source: NIH/NHLBI R01 HL107654

Abstract 238: Interleukin 17 Production Induced by Angiotensin Ii Type 2 Receptor Activation in T Cells Drives Flow-mediated Outward Remodeling of Mouse Resistance Arteries

Objective: Flow (shear stress)-mediated outward remodeling of resistance arteries is involved in collateral growth during postischemic revascularization. Besides the NO pathway the involvement of inflammatory factors and reactive oxygen species has been suggested in this remodeling although the mechanism and the sequence remains unknown. As T cells drive the inflammatory response, we investigated their role in flow-mediated remodeling. Mouse mesenteric resistance arteries (250μm internal diameter) were submitted in vivo to a chronic increase (144±18 to 239±25 μl/min) in blood flow. Arteries were collected for in vitro analysis after 1 to 7 days. Results: After 1 week, remodeling occurred in high flow (HF) arteries (diameter increased from 242±21μm to 324±20μm, n=9 per group, P&lt;00.1) in control mice. No remodeling occurred in nude mice and in mice treated with anti-CD3 antibodies. In control arteries lymphocyte T cells accumulate around HF arteries at day 1 (shown using both immunohistology and flow cytometry) and they expressed angiotensin II type 2 receptor (AT2R). In isolated T cells AT2R stimulation induced preferentially IL-17 production (20±6pg/ml to 50±5pg/ml, n=7 per group, P&lt;00.1). We then tested the occurrence of remodeling in mice lacking AT2R or IL-17. In both AT2R-/- and IL-17-/- mice diameter expansion did not occur whereas T cells and myeloid cells accumulation around the HF artery was not affected. IL-17 infusion (5ng/h, Alzet osmotic minipump) to nude mice and to AT2R-/- mice restored HF remodeling to control level. In order to confirm the role of AT2R in flow-mediated remodeling in another vascular territory, neovascularization was examined using an ischemic skin flap model in AT2R-/- mice. As in mesenteric arteries, the absence of AT2R prevented diameter expansion in the arteries feeding the skin flap. Conclusion: we demonstrate for the first time the involvement of lymphocytes T cells polarization into TH17 by angiotensin II type 2 receptor in flow-mediated outward remodeling of resistance arteries.

Heparin inhibits angiotensin II-induced vasoconstriction on isolated mouse mesenteric resistance arteries through Rho-A- and PKA-dependent pathways

Heparin is commonly used to treat intravascular thrombosis in children undergoing extracorporeal membrane oxygenation or cardiopulmonary bypass. These clinical circumstances are associated with elevated plasma levels of angiotensin II (Ang II). However, the mechanisms by which heparin modulates vascular reactivity of Ang II remain unclear. We hypothesized that heparin may offset Ang II-induced vasoconstriction on mesenteric resistance arteries through modulating the Rho-A/Rho kinase pathway. Vascular contractility was studied by using pressurized, resistance-sized mesenteric arteries from mice. Rho-A activation was measured by pull-down assay, and myosin light chain or PKA phosphorylation by immunoblotting. We found that heparin significantly attenuated vasoconstriction induced by Ang II but not that by KCl. The combined effect of Ang II with heparin was almost abolished by a specific Rho kinase inhibitor Y27632. Ang II stimulated Rho-A activation and myosin light chain phosphorylation, both responses were antagonized by heparin. Moreover, the inhibitory effect of heparin on Ang II-induced vasoconstriction was reversed by Rp-cAMPS (cAMP-dependent PKA inhibitor), blunted by ODQ (soluble guanylate cyclase inhibitor), and mimicked by a cell-permeable cGMP analogue, 8-Br-cGMP, but not by a cAMP analogue. PKC and Src kinase were not involved. We conclude that heparin inhibits Ang II-induced vasoconstriction through Rho-A/Rho kinase- and cGMP/PKA-dependent pathways.

Telmisartan inhibits vasoconstriction via PPARγ-dependent expression and activation of endothelial nitric oxide synthase

Telmisartan activates peroxisome proliferator-activated receptor-γ (PPARγ) in addition to serving as an angiotensin II type 1 receptor (AT(1)R) blocker. The PPARγ activity of telmisartan on resistance arteries has remained largely unknown. The present study investigated the hypothesis that telmisartan inhibited vascular tension in mouse mesenteric resistance arteries, which was attributed to an increased nitric oxide (NO) production through the PPARγ-dependent augmentation of expression and activity of endothelial nitric oxide synthase (eNOS). Second-order mesenteric arteries were isolated from male C57BL/6J, eNOS knockout and PPARγ knockout mice and changes in vascular tension were determined by isometric force measurement with a myograph. Expression and activation of relevant proteins were analysed by Western blotting. Real-time NO production was measured by confocal microscopy using the dye DAF. Telmisartan inhibited 9,11-dideoxy-11α,9α-epoxymethanoprostaglandin F(2α) (U46619)- or endothelin-1-induced contractions. An NOS inhibitor, N(G)-nitro-L-arginine methyl ester (l-NAME), or an inhibitor of soluble guanylate cyclase, 1H-[1,2,4]-oxadizolo[4,3-a]quinoxalin-1-one (ODQ), prevented telmisartan-induced inhibition of U46619 contractions. A PPARγ antagonist, GW9662, abolished telmisartan-induced inhibition. Likewise, the PPARγ antagonist rosiglitazone attenuated U46619-induced contractions. The effects of telmisartan and rosiglitazone were prevented by actinomycin-D, a transcription inhibitor. In contrast, losartan, olmesartan, and irbesartan did not inhibit contractions. The inhibition was absent in mesenteric arteries from eNOS knockout or PPARγ knockout mice. Telmisartan augmented eNOS expression, phosphorylation, and NO production, which were reversed by the co-treatment with GW9662. The present results suggest that telmisartan-induced inhibition of vasoconstriction in resistance arteries is mediated through a PPARγ-dependent increase in eNOS expression and activity that is unrelated to AT₁R blockade.

Epoxyeicosatrienoic Acids Function as Selective, Endogenous Antagonists of Native Thromboxane Receptors: Identification of a Novel Mechanism of Vasodilation

Epoxy- and dihydroxy-eicosatrienoic acids (EETs and DHETs) are vasoactive cytochrome P450 metabolites of arachidonic acid. Interestingly, however, the mechanism(s) by which EETs/DHETs mediate smooth muscle relaxation remains unclear. In contrast to previous reports, where dilation was purportedly large-conductance Ca(2+)-activated K(+) (BK(Ca)) and/or transient receptor potential cation channel, subfamily V, member 4 (TRPV4) channel-mediated, 14,15-EET-induced vasodilation [reversal of contractile tone established with the thromboxane receptor (TP) agonist 15-hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic acid (U-46619)] was unaltered in BK(Ca) and TRPV4 knockout mouse isolated aortae compared with wild-type controls, indicating a significant BK(Ca)/TRPV4-resistant mechanism. Whereas all EET and DHET regioisomers reversed U-46619 contraction in rat aortae and mouse mesenteric resistance arteries, these eicosanoids failed to alter phenylephrine-induced contraction, suggesting that they mediated dilation via a "TP-selective" mechanism. Competitive TP antagonism was also observed in nonvascular tissue, including rat fundus and tertiary bronchus, indicating that the effect is not specific to blood vessels. Such effects were TP-selective because 14,15-EET failed to inhibit "non-TP" prostanoid receptor-mediated function in multiple cell/tissue-based assays (K(b) > 10 microM). In accordance, 14,15-EET inhibited specific [(3)H]7-(3-((2-((phenylamino)carbonyl)hydrazino)-methyl)-7-oxabicyclo(2.2.1)hept-2-yl)-5-heptenoic acid (SQ-29548) binding to human recombinant TP receptor, with a K(i) value of 3.2 microM, and it showed weaker affinity for non-TP prostanoid receptors, including DP, FP, EP(1-4), and IP receptors (K(i) values of 6.1, 5.3, 42.6, 19.7, 13.2, 20.2, and >25 microM, respectively) and no appreciable affinity (K(i) values >10 microM) for a diverse array of pharmacologically distinct receptors, including the leukotriene receptors Cys-LT(1/2) and BLT(1). As such, EETs/DHETs represent a unique class of "endogenous" G protein-coupled receptor competitive antagonists, inducing vasodilation via direct TP inhibition. Thus, EETs/DHETs represent novel autoregulatory agents, directly modulating the actions of cyclooxygenase-derived eicosanoids following arachidonic acid mobilization.

Blood flow-dependent arterial remodelling is facilitated by inflammation but directed by vascular tone

Altered blood flow affects vascular tone, attracts inflammatory cells, and leads to microvascular remodelling. We tested the hypothesis that inflammation facilitates the remodelling response, but that vascular tone determines its direction (inward or outward). Mouse mesenteric resistance arteries were ligated to create either increased blood flow or low blood flow in vivo. In vivo microscopy was used to determine changes in vascular tone. Structural remodelling was studied after 2 days, with or without macrophage depletion. In order to characterize the inflammatory response, immunostaining, confocal microscopy, and real-time PCR were used. To address the role of vascular tone in remodelling, arteries were treated with the vasodilator amlodipine during organ culture. Vessels exposed to high blood flow dilated, whereas low flow induced constriction. After 1 day, inflammatory markers showed a complex but remarkably similar increase in expression during high flow and low flow. Both high-flow and low-flow vessels showed an increase in the number of adventitial macrophages. Depletion of macrophages eliminated flow-induced remodelling. Manipulation of vascular tone reversed inward remodelling in response to low blood flow. Altered blood flow triggers an inflammatory response that facilitates remodelling. Vascular tone is a crucial determinant of the direction of the remodelling response.

Extracellular Superoxide Dismutase Depletion in Hypertension Unmasks a New Role for Angiotensin II in Regulating Cu,Zn-Superoxide Dismutase Activity

While studying the effects of 2 weeks of angiotensin II infusion in extracellular superoxide dismutase knockout (ecSOD−/−) mice on increasing superoxide and promoting hypertension, Gongora et al1 made the surprising observation that NO production and its associated endothelium-dependent relaxation were actually improved by angiotensin II treatment in aorta from these mice without altering the expression of endothelial NO synthase. The studies were initially designed to further examine in ecSOD−/− mice the concept that an elevation of superoxide causes a compensatory increase in the expression of ecSOD, which blunts the hypertensive response to angiotensin II and preserves endothelium-dependent vasodilatation. Evidence for this initial hypothesis was seen in measurements of blood pressure and in the response of isolated mesenteric resistance arteries. Angiotensin II treatment was observed to cause the hypothesized greater increase in blood pressure in the ecSOD−/− mice compared with control animals and a loss of endothelium-dependent relaxation to acetylcholine, associated with the detection of increased superoxide in the resistance arteries. Endothelium-dependent relaxation to acetylcholine was impaired in the aorta of ecSOD−/− mice even in the absence of angiotensin II treatment. Thus, a dominant effect of depleted ecSOD in resistance arteries and aorta is an attenuation of endothelium-dependent relaxation, which could originate from superoxide scavenging NO and/or impairment of the conversion of superoxide to hydrogen peroxide, a potential alternative mediator of endothelium-dependent relaxation in mouse mesenteric resistance arteries.2 The surprising observation made in the study by Gongora …

Hydrogen peroxide acts as both vasodilator and vasoconstrictor in the control of perfused mouse mesenteric resistance arteries

Hydrogen peroxide (H2O2) plays a key role in the control of resistance artery (RA) tone and is hypothesized as an endothelial-derived hyperpolarizing factor. In this study, we investigated the effects of the same concentration of exogenous H2O2 on mouse mesenteric RA tone induced by intraluminal pressure, G-protein coupled receptor activation and KCl. RAs were cannulated in an arteriograph in the absence or presence of vasoconstrictors. RA developed myogenic tone (MT) in response to stepwise pressure increases. Under phenylephrine, H2O2 induced a dose-dependent (1-50 micromol/l) vasodilation with maximum dilation at 50 micromol/l. H2O2 at 50 micromol/l induced a full dilation of RA under MT or contraction by phenylephrine that was independent of nitric oxide synthase, cyclooxygenase, endothelium and potassium channels iberiotoxin and apamin sensitive. The Ca2+ channel inhibitor, nimodipine, significantly blocked MT and also the contraction to phenylephrine and KCl. Under these conditions, H2O2 had no effect on RA diameter. Under KCl, the same concentration of H2O2 induced a potent vasoconstriction. This contraction involved p38 mitogen-activated protein-kinase activation but not ERK1/2. These findings provide the first evidence showing that the same and low concentrations of H2O2 can act as a relaxing factor but also as a vasoconstrictor under conditions in which hyperpolarization is compromised.

Tumor necrosis factor increases the sensitivity of the contractile apparatus in isolated mouse resistance arteries via a calcium-dependent mechanism

The myogenic response is the modulation of resistance artery diameter in response to changes in luminal pressure. It is thought to be an important determinant of peripheral vascular resistance and therefore of arterial blood pressure. Since altered peripheral resistance is known to occur during the systemic inflammatory response syndrome (such as in sepsis), we suspect that an abnormal myogenic response might be responsible. Our main objective is to assess the effects of tumor necrosis factor alpha (TNF-α) on the myogenic response of mouse mesenteric resistance arteries and to determine the role of Ca2+ in this response.

Involvement of Metalloproteinases 2/9 in Epidermal Growth Factor Receptor Transactivation in Pressure-Induced Myogenic Tone in Mouse Mesenteric Resistance Arteries

Epidermal growth factor receptor (EGFR) transactivation is a mediator of angiotensin II (Ang II) signaling in cultured vascular smooth muscle cells isolated from large arteries. The present study used mouse mesenteric resistance arteries (MRAs) to investigate the role of EGFR transactivation under pressure-induced myogenic tone (MT). Isolated MRAs were mounted in an arteriograph and stimulated by 25 to 125 mm Hg or with Ang II and KCl. Stepwise increases in pressure resulted in MT development associated with increased EGFR phosphorylation and release of heparin-binding EGF (HB-EGF), a membrane-bound growth factor that is shed on cleavage by metalloproteinases. EGF (50 ng/mL) potentiated MT (59+/-1% to 51+/-0.6% of passive diameter at 75 mm Hg). Pretreatment with the EGFR inhibitors AG1478 (5 micromol/L) or PD153035 (1 micromol/L) significantly decreased MT. However, EGFR inhibitors had no effect on Ang II- and KCl-induced contraction. MT was potentiated by HB-EGF, 50 ng/mL, which is bound to the cell membrane and released on cleavage by metalloproteinases. Neutralizing HB-EGF antibodies or heparin treatment to sequester HB-EGF resulted in significant inhibition of pressure-induced MT. MT increased matrix metalloproteinase (MMP) 2 and MMP-9 gelatinase activity assessed by zymography, and specific MMP 2/9 inhibitors significantly decreased MT. These novel findings suggest that the mechanism of pressure-induced MT involves metalloproteinases 2/9 activation with subsequent HB-EGF release and EGFR transactivation.

Defect in microvascular adaptation to chronic changes in blood flow in mice lacking the gene encoding for dystrophin.

Dystrophin has a key role in striated muscle mechanotransduction. In mice lacking the gene encoding for dystrophin (mdx mice), the absence of dystrophin and several other proteins of the dystrophin-glycoprotein complex induces a defect in flow (shear stress)-mediated NO-dependent dilation (FMD). Because the endothelium is essential for the adaptation of arteries to chronic changes in blood flow, the long-term consequences of this vascular deficiency might affect flow-induced vascular remodeling. Thus, we submitted mouse mesenteric resistance arteries to chronic changes in flow by alternatively ligating arteries. Arteries were thus submitted to high flow (HF), low flow (LF), or normal flow. After 2 weeks, arteries were studied in vitro in an arteriograph. Increases in diameter (from 174+/-10 to 210+/-15 microm, pressure 75 mm Hg) found in HF arteries were not significant in mdx mice. Arterial diameters in LF arteries decreased similarly in control and mdx mice. FMD increased in HF arteries and decreased in LF arteries. FMD was not increased in HF arteries in mdx mice. NO-dependent FMD and NO synthase expression increased in the HF arteries of control mice but not in those of mdx mice. Dilatory and contractile tone, depending on the smooth muscle, was unaffected in HF arteries but decreased in LF arteries of both strains. We conclude that resistance arteries of mdx mice do not adapt properly to chronic changes in flow, inasmuch as the increases in diameter, endothelial NO synthase expression, and FMD did not occur in mdx mice submitted to HF for 2 weeks. This study suggests that blood flow regulation might be disturbed in dystrophin-related myopathies, possibly increasing organ damage.