Order Mesenteric Arteries Research Articles

Salvianolic acid B ameliorates vascular endothelial dysfunction through influencing a bone morphogenetic protein 4-ROS cycle in diabetic mice

AimThis study investigated the roles of bone morphogenetic protein-4 (BMP4) and ROS in diabetic endothelial dysfunction and explored whether Salvianolic acid B (Sal B) improved endothelial function by affecting BMP4-ROS in diabetic mice. Main methodsdb/db mice were orally administrated with Sal B (10 mg/kg/day) for one week while db/m + mice were injected with adenoviral vectors delivering BMP4 (3 × 108 pfu) and then received one week-Sal B treatment. ROS levels were assayed by DHE staining. Protein expression and phosphorylation were evaluated by Western blot. Aortic rings were suspended in myograph for force measurement. Flow-mediated dilatations in the second-order mesenteric arteries were determined by pressure myograph. Key findingsWe first revealed the existence of a BMP4-ROS cycle in db/db mice, which stimulated p38 MAPK/JNK/caspase 3 and thus participated in endothelial dysfunction. One week-treatment or 24 h-incubation with Sal B disrupted the cycle, suppressed p38 MAPK/JNK/caspase 3 cascade, and improved endothelium-dependent relaxations (EDRs) in db/db mouse aortas. Importantly, in vivo Sal B treatment also improved flow-mediated dilatation in db/db mouse second order mesenteric arteries. Furthermore, in vivo BMP4 overexpression induced oxidative stress, stimulated p38 MAPK/JNK/caspase 3, and impaired EDRs in db/m + mouse aortas, which were all reversed by Sal B. SignificanceThe present study demonstrates that Sal B ameliorates endothelial dysfunction through breaking the BMP4-ROS cycle and subsequently inhibiting p38 MAPK/JNK/caspase 3 in diabetic mice and provides evidence for the additional new mechanism underlying the benefit of Sal B against diabetic vasculopathy.

Activation of Nav channels by veratridine in mesenteric arteries: Contractile or relaxation responses?

Veratridine (VTD), a selective activator of voltage-gated Na + (Nav) channels, triggers contractile responses in mesenteric arteries (MA), by increasing intracellular Ca2+ mediated by Na + /Ca 2+ exchanger (NCX) in smooth muscle cells (SMC). However, VTD induces endothelium-dependent relaxation of retinal and cremaster arteries. These opposite VTD-responses gives rise to the question as to whether Nav channels play a role in vascular tone. We aim to characterize the contribution of Nav channels to vascular tone in MA. Since Nav channels are expressed in SMC, endothelial cells (EC) and nerve terminal endings of arteries, our aim is also to identify those which are responsible of VTD-evoked vasomotor responses. The VTD-induced contractile responses of first order mesenteric arteries (FOMA) and cecolic arteries (CA) of mice were recorded by wire myography. EC were used to characterize Ca 2+ responses using FURA-2 probe. Western blot and absolute RT-qPCR were used to characterize Nav channels and NCX expression. Nav1.2 channels are expressed in both SMC and EC of MA. VTD elicits contraction of FOMA, which is abolished by prazosin. Surprisingly, VTD induces relaxation of CA. This vasorelaxation is resistant to prazosin and supressed by atropine. VTD-induced relaxation is inhibited by eNO-synthase inhibitor (L-NNA) and NCX antagonists. Morever, NCX1 is the main NCX isoform expressed in EC. Finally, acetylcholine-induced Ca 2+ reponses are inhibited by NCX antagonists in endothelial cells. VTD induces opposite contractile responses in isolated mesenteric arteries. In fact, VTD activates Nav channels of sympathetic neurons in FOMA, and parasympathetic neurons in CA, leading to noradrenaline and acetylcholine release, respectively. Thus, the vascular tone of both arteries are differently regulated by the autonomic nervous system. Finally, acetylcholine triggers Ca 2+ -activation of eNO-synthase mediated by NCX1 in CA.

Endothelial RIPK3 Regulation and its Effects on Vascular Permeability Following Intestinal Ischemia‐Reperfusion Injury

The cytoplasmic protein receptor-interacting serine/threonine-protein kinase 3 (RIPK3) is primarily known for promoting necroptosis, but more recent findings suggest that RIPK3 has additional functions separate from necroptosis. Our lab recently demonstrated that in the absence of the chromatin-remodeling enzyme CHD4, RIPK3 is elevated in hypoxic cultured embryonic endothelial cells (ECs) and that elevated RIPK3 causes vessel fragility in midgestation mouse embryos. Our current study investigates the regulation of endothelial RIPK3 following intestinal ischemia-reperfusion injury (IRI) in mice and its effects on vascular permeability and damage following IRI. To assess RIPK3 regulation following IRI, first-order branches of the superior mesenteric artery were clamped for 1 hr to induce ischemia, and clamps were removed subsequently for 24 hr reperfusion (C57BL/6J mice; 10-12 weeks old). Unclamped vessels and downstream gut tissue from the same animal were used as sham controls. RIPK3 and VE-Cadherin expression (mean fluorescence intensity; MFI) were quantified in 1st and 2nd order mesenteric arteries using immunostaining of en face vessel preparations. RIPK3 was significantly elevated (P=0.0216) at 1 hr ischemia and following 24 hr IR injury (P=0.0295) compared to sham controls (7-8 vessels/group, n=4 mice). Junctional VE-Cadherin showed increased expression (not significant) following 1 hr ischemia. Although no changes were observed following 24 hr IRI, VE-Cadherin expression at mesenteric arterial endothelial junctions were discontinuous and unstable suggesting a potential barrier dysfunction associated with IRI. To assess the role of RIPK3 on vascular permeability, we used Ripk3fl/fl;Cdh5(PAC)-CreERT2 mice (Ripk3 endothelial cell knockout=RIPK3ECKO) compared to littermate controls, and vascular permeability was assessed using the Evans blue assay following 24 hr intestinal IRI. Gut vascular permeability was increased following IRI in both RIPK3ECKO (n=4 mice)and littermate controls (n=3 mice), however permeability was also significantly increased (P=0.02) in sham gut regions and the livers (P=0.04) of RIPK3ECKO mice unlike in controls. Collectively, these data suggest that deletion of endothelial RIPK3 increases vascular damage downstream of injury sites and points toward a protective role for RIPK3 in the vasculature following IRI. Future studies evaluating mechanisms of RIPK3 protective effects in endothelial cells following IRI will provide insight into therapeutic targets for vessel recovery from IRI.

IL‐10 Neither Causes Direct Vasorelaxation Nor Reduces Vasoconstriction in Rat Mesenteric Resistance Arteries

Perivascular adipose tissue (PVAT) is the fat that surrounds most of the peripheral blood vessels in the body. PVAT is protective and reduces contraction of blood vessels in health. We discovered that in health, PVAT around the rat small mesenteric resistance vessels (MRPVAT) contained a community of immune cells including T cells, B cells, macrophages, NK cells, neutrophils and mast cells. Interleukin (IL)‐10‐‐an anti‐inflammatory cytokine usually produced by T cells, B cells and macrophages‐‐was identified as one of the highly expressed (mRNA) cytokines in MRPVAT. One report suggests that IL‐10 causes relaxation of mouse mesenteric arteries. Hence, we hypothesized that IL‐10 directly causes vasorelaxation and/or reduces vasoconstriction in healthy rats. Male Sprague Dawley rats (12–14 weeks age) were used. Mesenteric resistance arteries expressed the receptor for IL‐10 (tunica intima and media) as determined by immunohistochemistry. Isolated tissue bath and third order mesenteric resistance arteries (without PVAT and with intact endothelium) were used for isometric contractility studies. Increasing concentrations [0.4–100 ng/mL] of recombinant rat (rr) IL‐10 or vehicle was directly added to half‐maximally constricted (phenylephrine PE or thromboxane A2 agonist U46619) vessels. IL‐10 did not reduce contraction, suggesting it was not a direct vasorelaxant. Further, the ability of rrIL‐10 to cause a rightward or downward shift of a vasoconstrictant‐response curve was tested. The vessels were incubated with rrIL‐10 [100 ng/mL or 10 ng/mL] or vehicle for 1.5 hours in the tissue bath followed by a cumulative PE [10−9–10−5 M] or U46619 [10−10–10−5 M] response curve. The maximal contractions or EC50 values (shown in table ) were not statistically different in rrIL‐10 incubated vessels vs vehicle. Thus, acute exposure of exogenous IL‐10 neither caused direct vasorelaxation nor reduced vasoconstriction. Further studies with neutralizing antibody for IL‐10 to vessels ± PVAT will help determine if IL‐10 produced in the PVAT causes vasorelaxation and/or reduction in local vasoconstriction indirectly, thus contributing to the anti‐contractile nature of PVAT.Support or Funding InformationNIH HL70687 −Log EC50 ± SEM (n=6–10 arteries/3–5 rats/curve) Vehicle rrIL‐10 (100 ng/mL) PE curve 5.7± 0.05 5.6±0.05 U46619 curve 7.6±0.28 7.2±0.59 Vehicle rrIL‐10 (10 ng/mL) PE curve 5.7±0.03 5.6±0.04 U46619 curve 7.1±0.12 7.2±0.13

Quantitatively defining spatial patterns of myoendothelial junctions in the arterial wall

The regulation of vasoconstriction and vasodilatory pathways is essential in resistance arteries, and can be modulated by heterocellular communication between the endothelial and smooth muscle cells. This can occur via direct endothelial‐smooth muscle cell contact at myoendothelial junctions (MEJs) through fenestrations in the internal elastic lamina (IEL). While it has been demonstrated that MEJs do not occupy every fenestration, their spatial distribution throughout the endothelium has not yet been defined and could reveal localization tendencies to cellular landmarks. We hypothesized that the MEJ and IEL fenestration spatial distribution would exhibit a non‐random distribution pattern throughout the endothelium with respect to various cellular landmarks, including the interendothelial junctions. In order to quantify this relationship, the spatial distributions of both holes and MEJs with respect to various cellular landmarks were examined. Third order mesenteric resistance arteries isolated from C57Bl6 male mice were prepared en face to view the endothelium and detect the IEL, nuclei, myoendothelial junctions, and interendothelial junctions (e.g., claudin‐5). A Matlab based analysis pipeline was developed to calculate the proximity of IEL fenestrations to cellular landmarks, such as interendothelial junctions. En face views of the IEL often have patches of varying signal intensity, posing a challenge for detecting fenestrations through basic thresholding methods. To overcome this, parameters within Image Processing Toolbox documentation were optimized for each image prior to thresholding. This allowed for accurate assignment of XY coordinates to fenestration boundaries (bwboundaries documentation) and calculation of center points. En face views of claudin‐5 were similarly thresholded and their boundaries also defined by XY coordinate matrix. Next, Euclidean distance was calculated between each fenestration center point and all claudin‐5 XY coordinates and plotted as a probability distribution. Monte Carlo simulations were also performed as a negative control. In the simulation, the fenestrations are replaced by randomly generated circles that mimic the number and radii of fenestrations in the original image. This is compared to the original distribution to assess if identified patterns are random. Our analysis of >100 simulations indicate that IEL fenestrations appear to be randomly distributed across the IEL. However, we find the MEJs within the IEL fenestrations may be strategically placed near interendothelial junctions. Preliminary data indicates that this particular MEJ localization pattern may be opportunistic because of the cytoskeletal components at interendothelial junction that can be recruited to support the signaling microdomains within MEJs.Support or Funding InformationThis work supported by 1F31HL149228‐01 (CAR) and HL088554 (BEI).

IP3‐evoked Ca2+ release in Endothelial Cells is Modulated by the Immunosuppressant FK506‐binding Protein (FKBP12)

The endoplasmic reticulum (ER) is integral to the regulation of cytoplasmic Ca2+ concentration in endothelial cells. Ca2+ release from the ER is controlled by two intracellular receptor/channel complexes, the inositol 1,4,5‐triphosphate receptor (IP3R) and the ryanodine receptor (RyR). These receptors have been shown to be regulated by the accessory FK506‐binding protein (FKBP). FK506 is a potent immunosuppressant, which is routinely used after organ transplant to lower the risk of rejection, and has also been used in drug‐eluting stents. FK506 has been shown to act on smooth muscle cells either directly by binding to the channel, or indirectly via FKBP modulation of two targets, calcineurin or the mammalian target of rapamycin (mTOR). However very little is known on the role FKBPs play in endothelial cell Ca2+ signaling.To investigate this, Ca2+ signals were measured in the endothelium of second order mesenteric arteries using Cal‐520 (5 μM). IP3Rs were activated by localised photo‐uncaging of IP3 (5 μM), and RyR by the application of Caffeine (10 mM). FK506 (10 μM), which displaces FKBP from each receptor to inhibit calceinurin, increased the IP3‐evoked Ca2+ rise. The phosphatase inhibitors cypermethrin and okadaic acid also increased the IP3‐mediated Ca2+ release. Cypermethrin and okadaic acid did not prevent FK506‐induced increase in Ca2+ release. These results suggest that while calceinurin may modulate IP3‐evoked Ca2+ release, the phosphatase is not necessary for the FK506‐mediated potentiation of IP3‐evoked Ca2+ increase.Rapamycin (10 μM), which disrupts the binding of FKBP12 to the receptor and inhibits mTOR, did not significantly alter IP3‐induced Ca2+ release in endothelial cells, indicating that mTOR does not exert a significant role in endothelial IP3‐induced Ca2+ release.Interestingly Caffeine, the activator of RyR‐mediated Ca2+ release, did not activate Ca2+ signals in the endothelium either in intact tissue or in freshly isolated endothelial patches, suggesting that Caffeine‐activated RyRs do not play a role in endothelial Ca2+ release.These results indicate that FKBPs play an integral role in regulating endothelial IP3‐mediated Ca2+ release, and may underlie the severe endothelial dysfunction associated with FK506 immunosuppressants.Support or Funding InformationWellcome TrustBritish Heart FoundationThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Endothelial Heterogeneity Enables Parallel Processing of Multiple Stimuli

The endothelium is the innermost layer of all blood vessels and changes in the behavior of this single layer of cells underlie almost all cardiovascular disease. To regulate cardiovascular function, the endothelium must integrate hundreds of signals. These signals can arrive from as close as neighboring cells to substances circulating in the blood from the most remote outpost of the body. However, how the endothelium detects and relays the information stored in each of these signals to initiate a physiological output is not fully understood. The problem in integrating signals is made even more complex as the central relay system within an endothelial cell is a single ion, Ca2+. Here, we show the endothelium manages a multitude of extracellular signals by utilizing spatially‐distinct endothelial cells. Furthermore, the Ca2+ signal generate by each activator has unique distinguishing characteristics.To determine how multiple extracellular signals are detected in the endothelium we analysed the Ca2+ activity from ~1000 endothelial cells from intact arteries. Male‐Sprague‐Dawley rats were euthanized by CO2 overdose and second order mesenteric arteries were extracted, cut open and pinned flat. The endothelium was loaded with a Ca2+ indicator Cal‐520/AM and imaged using fluorescence microscopy. Ca2+ activity was separately analysed in each of the 1000 individual endothelial cells using a custom built software package written in Python.For each activator (ACh, ATP, ADP & Histamine) full concentration response curves were carried out to determine the EC25 concentration (the concentration at which 25% of cells responded). The addition of the four activators (each separately applied), at their respective EC25 concentration, to the same field of endothelium revealed the endothelium utilizes sub‐populations of endothelial cells to detect each activator. The Ca2+ signal began in small sub‐populations of endothelial cells that were organised in clusters and propagated between cells as Ca2+ waves. The cells responsible for the detection of a specific activator were spatially unique and there was no significant overlap with cells that were primed to detect another agonist (P<0.05). Each activator generated a unique Ca2+ signal that was specific to the agonist. When the activators were applied in combination at the EC25 concentration, the unique characteristics of the Ca2+ signal for a specific activator when applied alone was drastically altered. Interestingly, the characteristics of the Ca2+ signal when applied in combination was not a simple linear summation of the individual signals. Expansive responses occurred, in that the outputs exceeded the sum of the inputs. These results suggest individual endothelial cells communicate with their neighbors and complex computations are carried out by combining the information from each source to generate a distinct output. By using spatially‐distinct cells, unique signal characteristics and communication, the endothelium is able to detect and relay the information stored in the multitude of extracellular activators to initiate physiological outputs.Support or Funding InformationWellcome Trust & British Heart FoundationThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Arterial smooth muscle cell PKD2 (TRPP1) channels contribute to angiotensin‐induced vascular remodeling

Hypertension is associated with structural changes of resistance‐sized arteries, a process referred to as vascular remodeling, resulting in an increase in total peripheral resistance. One stimulus that exerts changes in arterial structure is an elevation in the vasoconstrictor angiotensin II (ang II), which mediates vascular smooth muscle cell growth, consequently resulting in vascular remodeling. Arterial smooth muscle cell PKD2 (TRPP1) channels, a member of the transient receptor potential (TRP) channel superfamily of ion channels, are upregulated in resistance‐sized arteries in ang II‐treated mice when compared to saline‐perfused controls. In addition, ang II‐treated conditional smooth muscle‐specific PKD2 knockout (PKD2sm−/−) mice demonstrate attenuated arterial wall remodeling and reduced blood pressure. An alteration in the expression and function of vascular smooth muscle cell ion channels also contributes to vascular remodeling through the promotion of cell proliferation. Here, we demonstrate that transient receptor potential canonical type 3 (TRPC3) and epidermal growth factor receptor (EGFR) channels, both indicated previously to contribute to ang II‐mediated vascular remodeling, are upregulated in mesenteric arteries isolated from ang II‐treated mice. During hypertension, TRPC3 channel function is also elevated, as demonstrated by application of the TRPC3 channel blocker pyrazole 3 (Pyr3) to pressurized 3rd and 4th order mesenteric arteries. Ang II‐treated PKD2sm−/− mice show reduced TRPC3 and EGFR expression, as well as decreased TRPC3 function when compared to ang II‐treated PKD2fl/fl controls. These data suggest that arterial smooth muscle cell PKD2 channels may contribute to TRPC3‐induced vascular remodeling in resistance‐sized mesenteric arteries.Support or Funding InformationAHA 16SDG27460007This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Extracellular vesicles from Wistar Kyoto and spontaneously hypertensive rats have differential vasodilatory responses in resistance arteries

Extracellular vesicles (EVs) have been described as novel bio‐markers and bio‐activators in vascular dysfunction in hypertension (HTN). However, the mechanism by which EVs affect vascular function is unknown. To examine the effects of EVs on acetylcholine (ACh)‐mediated vasodilation, we isolated 3rd order mesenteric arteries and EVs from 12‐wk old normotensive Wistar‐Kyoto (WKY) and spontaneously hypertensive (SHR) rats. Arteries were cannulated on a pressure myograph and EVs (~6×107EV/mL) were added to the vessel lumen and circulating bath. No significant difference in ACh vasodilation was observed in control WKY and SHR arteries (no EVs). Interestingly, WKY arteries treated with WKY EVs reduced vasodilation compared to control arteries, an effect lost when treated with SHR EVs. ACh‐mediated vasodilation was not altered in SHR arteries when treated with either WKY or SHR EVs. WKY arteries treated with EVs isolated from 6‐wk old WKY and SHR, an age where SHR rats have not yet developed HTN, or with delipidated EVs from 12‐wk old WKY and SHR rats resulted in reduced vasodilation. Together, these data suggest that normotensive EVs reduce ACh‐mediated vasodilation and upon development of hypertension fail to reduced vasodilation. However, reduced vasodilation can be restored by delipidation of the SHR EVs or SHR EVs obtained prior to development of hypertension. Analysis of isolated EVs showed a larger number of CD45‐positive EVs in 12 week‐old SHR rats, indicating an increase in EVs from inflammatory cells following the development of hypertension. Using EVs isolated from human blood samples, a similar reduction in vasodilation was observed in arteries exposed to EVs isolated from normotensive humans. This effect was lost when arteries were treated with EVs isolated from hypertensive patients. This data supports the functional role of EVs in vascular regulation that is altered in HTN.Support or Funding InformationK99HL143165, K23HL12610, HL088554This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Parallel Processing of Multiple Stimuli in the Vascular Endothelium

The endothelium is a complex network of cells that lines the entire vasculature and controls virtually all cardiovascular functions. Changes in the behaviour of endothelial cells underlies almost all cardiovascular disease. To regulate cardiovascular function, the endothelium integrates hundreds of signals that provide constant instructions. Signals arrive from as close as neighbouring endothelial cells and underlying smooth muscle cells to substances circulating from the most remote outpost of the body. These signals provide endless streams of information to the endothelium that must be integrated and decoded. How this is achieved is not understood. Here, we show the endothelium manages a multitude of extracellular signals by using spatially‐distinct endothelial cells that are primed to detect specific extracellular signals. In response to each extracellular signal, cells generate intracellular messages with unique characteristics. When multiple extracellular signals are present messages are communicated across cells and computations carried out to generate a consensus. These endothelial mechanisms may underlie how signals that utilise only one messenger (Ca2+) can generate different stimulus‐specific responses across a vast number of cells.To determine how signals evoked by multiple agonists (ACh, ATP, ADP & histamine) were transduced in the endothelium, we studied Ca2+ signals evoked by the extracellular activators in intact arteries. Male Sprague‐Dawley rats were euthanized by CO2 overdose and second order mesenteric arteries were extracted, cut open and pinned flat. The exposed endothelium was loaded with a Ca2+ indicator, Cal‐520/AM and imaged using fluorescence microscopy. Changes in intracellular Ca2+ in thousands of endothelial cells were measured simultaneously and a custom analysis package written in Python was used to analyse the data.For each agonist, full concentration response curves were carried out to determine the EC25 concentrations (the concentration at which 25% of the cells were activated). Interestingly, the EC25 concentration increased 10‐fold between ACh (7.5 nM) & ADP (61.1 nM) and 100 fold between ACh & ATP (627.1 nM) and also for ACh & histamine (1.1 μM). The Ca2+ signal to the EC25 for each agonist began in spatially‐distinct clusters and propagated between cells as Ca2+ waves. There was no significant overlap in the cells that first responded to each agonist or indeed in the entire population response (P<0.05). The activation of spatially‐distinct cells permits the endothelium to selectively process separate information and functions in parallel. A critical aspect of endothelial transduction of extracellular signals is how a single ion can control a host of different responses. Previous studies have shown that changes in the amplitude, frequency and kinetics of a Ca2+ signal within a cell can lead to the generation of different responses. By studying thousands of endothelial cells our study reveals another novel feature of Ca2+ signaling. Each agonist, when applied at low concentrations (EC25) evoked unique Ca2+ signals. This may provide insight into how the response that is generated to different agonists is communicated between cells to give an endothelium wide response. These results suggest that spatially‐distinct, stimulus‐specific cells unravel the constant streams of information to the endothelium held in the multitude of extracellular signals.Support or Funding InformationThis work was supported by the Wellcome Trust and British Heart FoundationThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Nitroxyl Anion Mediates Relaxation in Mesenteric Arteries from Angiotensin II Hypertensive Mice.

Nitroxyl anion (HNO) has recently become an emerging candidate in vascular regulation. NO- is a potent vasodilator of both conduit and small resistance vessels and mediates relaxation in a soluble guanylate cyclase-dependent manner. Interestingly, HNO activates voltage-dependent K+ (K+ V) channels, whereas Nitric Oxide (NO) activates calcium-activated K+ Ca channels. To date, there are few studies investigating the role of HNO in hypertension, and the possible mechanisms, which may be altered during this condition. We hypothesized that mesenteric arteries from angiotensin II-induced (AngII) hypertensive mice would exhibit an increased dependence upon NO- for relaxation, which may be mediated through K+ V channels. Methods and Key Results: C57/Bl6 mice, aged 12-14 weeks were implanted with mini-pumps containing angiotensin II (AngII, 3600ng/kg/min) for 14 days. For this study, we proposed to investigate the role of HNO in the resistance vasculature, and so first order mesenteric arteries were isolated and used in functional studies, or were frozen for Western blot analysis. We observed that mesenteric arteries from AngII mice (AngII) exhibited a decrease in HNO-mediated relaxation, which was endotheliumindependent. With HNO scavenging by L-cysteine [3mM], the maximal acetylcholine (ACh)-mediated relaxation response was decreased in sham, whereas mesenteric arteries from AngII exhibited a decrease in sensitivity. Incubation with the K+ V channel inhibitor, 4-aminopyridine [1mM], decreased AChmediated relaxation responses in sham, but almost completely abolished relaxation in AngII. We reveal that exogenous HNO-mediated relaxation, via Angeli's Salt, is impaired in mesenteric arteries from AngII-treated mice, yet endogenous HNO-mediated relaxation may be more important during hypertension.

Abstract 167: Inhibition of Endoplasmic Reticulum Stress Improves Vascular Function in Type II Diabetic Mice

Cardiovascular disease is the leading cause of death in the United States with type 2 diabetes (T2D) representing a major risk factor in its development. Vascular dysfunction, characterized by arterial stiffness and endothelial dysfunction occur prior to overt cardiovascular disease and predict future cardiovascular events and mortality in diabetic individuals. Dysfunction of the endoplasmic reticulum (ER), or ER stress, is associated with the development and progression of chronic metabolic diseases such as obesity and T2D. However, the role of ER stress in the development of vascular dysfunction observed in T2D is unclear. We hypothesized that inhibiting ER stress would improve both measures of vascular dysfunction observed in diabetic mice. Male C57BL/6J Leprdb (DB) male mice lacking the leptin receptor were used as a model of T2D. Starting at 4 months of age DB mice were given intraperitoneal injections of ER stress inhibitor, tauroursodeoxycholic acid (TUDCA) at 250mg/kg/day for 4 weeks. C57BL/6J mice were used as controls (n=8 for all groups). Pulse wave velocity (PWV) was measured at baseline (prior to treatment) and after TUDCA treatment. Secondary order mesenteric resistance arteries (MRA) were used to determine endothelial dependent dilation (EDD). DB mice not treated with TUDCA were used for acute studies of EDD by incubating the MRA for 1hr with 0.5mM TUDCA. At baseline, DB mice displayed increased arterial stiffness compared to C57BL/6J controls as measured by PWV (457±25 vs 348±26 cm/s, p<0.05). TUDCA treatment significantly decreased PWV (402±12 cm/s, p<0.05). Both acute and chronic TUDCA treatment improved DB EDD compared to untreated controls (58.4±13.8 and 49.1±6.1%, vs 19.8±9.6%, p<0.05). Expression of ER stress-associated genes CHOP and ATF4 were significantly elevated in DB perivascular adipose tissue and treatment with TUDCA significantly decreased ATF4 compared to DB. In the aorta, TUDCA significantly decreased GRP78 and XBP1 compared to DB. These data support the hypothesis that ER stress contributes to the vascular dysfunction observed in T2D and suggest that ER stress may be a potential target in the treatment of T2D related vascular stiffening and dysfunction.

Hypercholesterolemia Induces Endothelial Dysfunction Through Impaired Kir2.1 Channel Activity Upstream of NO Production

Hypercholesterolemia‐induced endothelial dysfunction is largely attributed to a loss of nitric oxide production/release, however the underlying mechanisms are unclear. Our recent study demonstrated that endothelial inwardly‐rectifying K+ channels (Kir2.1) are essential for flow‐induced activation of eNOS, NO production and flow‐induced vasodilation (FIV) in murine mesenteric resistance arteries. Our group also showed earlier that Kir channels are strongly suppressed by the elevation of cellular cholesterol. Therefore, we hypothesize that under hypercholesterolemic conditions cholesterol‐induced inhibition of Kir2.1, the major mesenteric EC Kir channel, would result in endothelial dysfunction (i.e. reduced dilations to flow) ultimately by preventing NO production. To test this hypothesis, we first determined the impact of clinically relevant concentrations of LDL on Kir current in human aortic ECs. We show that at concentrations considered to be at risk for atherosclerosis and heart disease we observed decreased Kir current density (10.8 ± 1.0 pA/pF for Control vs. 5.1 ± 0.3 pA/pF for 150 mg/dL LDL vs. 2.5 ± 0.4 pA/pF for 250 mg/dL LDL), an effect significantly reversed by the presence of HDL (5.1 ± 0.7 pA/pF for 150 mg/dL LDL vs. 10.2 ± 2.3 pA/pF for 50 mg/dL HDL + LDL). We also show that while Kir currents in endothelial cells freshly‐isolated from mesenteric arteries of WT mice are clearly shear‐stress sensitive, sensitivity of the channels to shear is lost in dyslipidemic ApoE−/− mice. Furthermore, the loss of FIV in pressurized and preconstricted 1st order mesenteric arteries (100–150 μm lumen diameters) isolated from dyslipidemic ApoE−/− mice can be attributed to the loss of endothelial Kir function. Specifically, in WT mice exposure to an intralumenal shear stress of ~30 dyn/cm2 results in an average dilation of 93.1% of the maximal diameter while ApoE−/− mice exhibit dilations of only 36.4%. In Kir2.1‐deficient mice (Kir2.1+/−), maximal dilation is reached at 42.2% and no further decrease is observed in ApoE−/−/Kir2.1+/− double knock‐out mice suggesting that impaired Kir channels are responsible for the decrease in FIV in hypercholesterolemic conditions. Consistent with these observations, Ba2+, a Kir channel blocker, can reduce dilations to flow in arteries of WT mice but has little to no effect in arteries from ApoE−/− mice. Additionally, NO production in arteries of ApoE−/− and ApoE−/− × Kir2.1+/− mice exposed to internal flow for 30 minutes is decreased compared to WT arteries. Most importantly, we found that overexpressing Kir2.1 specifically in mesenteric endothelium using adenoviral transfection driven by the VE‐Cadherin promoter recovered both FMV and flow‐induced NO production in ApoE−/− or ApoE−/− × Kir2.1+/− arteries strongly confirming the role for EC Kir2.1 impairment in hypercholesterolemia.Support or Funding InformationNIH RO1 HL073965American Heart Association Postdoctoral Award 16POST27000011

Abstract 132: Adrenergic Receptor Blockade Prevents Placental Ischemia-induced Hypertension

Hypertensive disorders of pregnancy are the number one cause of pregnancy-related deaths in the United States. Preeclampsia is a disorder of maternal hypertension and cardiovascular dysfunction typically presenting in the second half of pregnancy along with fetal growth restriction. There are no steadfast therapies besides early delivery of the fetus and ischemic placenta, which releases factors into the maternal circulation promoting hypertension. Although sympathetic nervous activity was found to be increased in preeclamptic versus normal pregnant women, it is unknown if sympathetic nervous system plays a role in placental ischemia-induced hypertension. To address this question, we tested the hypothesis that adrenergic receptor blockade prevents placental ischemia-induced hypertension. Wistar rats were randomized to receive reduced uterine perfusion pressure, (RUPP, n=6) or Sham (n=5) surgeries on gestational day 14 and examined at day 19. In RUPP vs Sham rats, respectively, mean arterial blood pressure (115 ± 4 vs 103 ±2 mmHg, P<0.05) and the number of absorbed fetuses (6 ± 1 vs 1 ± 1, P<0.05) were greater whereas average fetal weight was lower (1.7 ± 0.1 vs 2.0 ± 0.2, P<0.05) with similar placental weights (0.49 ± 0.03 vs 0.52 ± 0.03). In RUPP vs Sham rats, renal cortical norepinephrine content (HPLC) was higher (183 ± 15 vs 150 ± 8 pg/mg wet weight, P<0.05) and vasoconstriction to phenylephrine was greater in small, third order mesenteric arteries (Emax: 262 ± 19 vs 160 ± 26% of KCl response). A subset of RUPP rats (n=3) received terazosin and propranolol (3 mg/kg per day each, subcutaneous osmotic minipump) to block alpha- and beta-adrenergic receptors, respectively, beginning the day of RUPP surgery. At day 19, adrenergic blockade prevented the development of hypertension (100 ± 4 mmHg, P<0.05) and did not alter number of fetal absorptions (8 ± 1). Average fetal weight was higher (2 ± 0.1, P<0.05) and placental weight lower (0.41 ± 0.03, P<0.05) compared to the untreated RUPP rats. In conclusion, placental ischemia-induced hypertension depends on activation of the sympathetic nervous system. The mechanism for this enhanced sympathetic nerve activity is unknown but may involve factors released from the ischemic placenta.

OP.8C.04] THE FLOW DETECTION ROLE OF THE VASCULAR EPITHELIAL SODIUM CHANNEL (ENAC) IN AN EXPERIMENTAL MODEL OF HYPERTENSION

Objective: Hypertension (HTN) impairs the regulation of blood flow leading to cardiovascular morbidity and mortality. A “new player” in blood-flow regulation is the endothelium Epithelial Sodium Channel (vascular ENaC). We hypothesise that dysfunction in HTN is due to changes in vascular ENaC activity. Aldosterone regulates renal ENaC and has a pivotal role in HTN and recently it has been suggested that aldosterone antagonism (spironolactone therapy) is beneficial beyond that predicted by the reduction in blood pressure. We propose that these additional effects are due to re-establishment of normal vascular function through modulation of vascular ENaC activity. Design and method: An experimental model of HTN, compared to normotensive (NTN) controls, with/without spironolactone therapy (n = 3–5) was used. HTN was induced in adult male transgenic rats (TG strain: Cyp1a1-Ren2), via diet-driven (0.167% w/w indol-3-carbinol) activation of renin-angiotensin-aldosterone system. Spironolactone treatment was administered daily (orally, 8.8 mg/kg/day) starting 7 weeks following initiation of HTN. Flow-mediated changes in the internal diameter (% change from no-flow) of isolated 3rd order mesenteric arteries, in the presence/absence of 10 μM amiloride were measured to determine the role of ENaC. Flow was induced through the vessels, whilst mean intraluminal pressure was maintained at 60 mmHg. Results: Isolated NTN arteries demonstrated flow mediated-constriction (−9.9 ± 3.6%) under basal conditions which was reduced (−6.3 ± 3.1%) by amiloride. The basal response was absent in HTN (0.8 ± 1.0%) vessels, and unaffected by amiloride (0 ± 1.5%). Spironolactone treatment of NTN rats resulted in a slight augmentation (−11.8 ± 1.9 %) of flow-mediated constriction which was unaffected by amiloride (−11.6 ± 1.7%). Spironolactone treatment did not improve the lack of flow-mediated response of HTN vessels (0.5 ± 0.7%), and was unaffected by amiloride (−0.4 ± 1.3%). Conclusions: Vascular ENaC activity augments flow-mediated constriction of mesenteric arteries. The attenuation of flow-mediated responsiveness in experimental RAAS-driven HTN is linked, in part, to a reduction in vascular ENaC activity. Spironolactone augments flow-mediated responsiveness of NTN vessels, although this may not be through increased ENaC activity. Initiation of spironolactone treatment at a late stage of HTN doesn’t appear to modulate ENaC function or re-establish normal vascular function.

Aging Reduces L-Type Calcium Channel Current and the Vasodilatory Response of Small Mesenteric Arteries to Calcium Channel Blockers.

Calcium channel blockers (CCBs) are widely used to treat cardiovascular disease (CVD) including hypertension. As aging is an independent risk factor for CVD, the use of CCBs increases with increasing age. Hence, this study was designed to evaluate the effect of aging on the sensitivity of small mesenteric arteries to L-type voltage-gated calcium channel (LTCC) blockers and also to investigate whether there was a concomitant change in calcium current density. Third order mesenteric arteries from male F344 rats, aged 2.5–3 months (young) and 22–26 months (old) were mounted on wire myograph to measure the tension during isometric contraction. Arteries were contracted with 100 mM KCl and were then relaxed in a cumulative concentration-response dependent manner with nifedipine (0.1 nM–1 μM), verapamil (0.1 nM–10 μM), or diltiazem (0.1 nM–10 μM). Relaxation-concentration response curves produced by cumulative concentrations of three different CCBs in arteries of old rats were shifted to the right with statistically significant IC50s. pIC50 ± s.e.m: (8.37 ± 0.06 vs. 8.04 ± 0.05, 7.40 ± 0.07 vs. 6.81 ± 0.04, and 6.58 ± 0.07 vs. 6.34 ± 0.06) in young vs. old. It was observed that the maximal contractions induced by phenylephrine and reversed by sodium nitroprusside were not different between young and old groups. However, Bay K 8644 (1 μM) increased resting tension by 23 ± 4.8% in young arteries and 4.7 ± 1.6% in old arteries. LTCC current density were also significantly lower in old arteries (−2.77 ± 0.45 pA/pF) compared to young arteries (−4.5 ± 0.40 pA/pF); with similar steady-state activation and inactivation curves. Parallel to this reduction, the expression of Cav1.2 protein was reduced by 57 ± 5% in arteries from old rats compared to those from young rats. In conclusion, our results suggest that aging reduces the response of small mesenteric arteries to the vasodilatory effect of the CCBs and this may be due to, at least in part, reduced current density of LTCC.

Defining the Mechanism of Erythropoietin's Vasoactive Properties in Resistance Arteries

Clinically, erythropoietin (EPO) is known to increase systemic vascular resistance, however, in cell culture, EPO has been reported to increase activation of endothelial nitric oxide synthase (eNOS). How EPO can activate eNOS, while causing vasoconstriction, is likely due to EPO's ability to activate multiple receptors, the homodimeric EPO receptor (EPOR2) and the heterodimeric EPOR/beta common receptor (βCR). The purpose of this study was to investigate the contribution of each receptor in endothelial‐dependent vasodilation. We hypothesized that the vasoconstrictive effects seen with EPO administration are mediated through the EPOR2. To study the effects of each receptor, we utilized EPO and the EPO peptide (EPOp), which solely acts on the heterodimeric EPOR/βCR. Adult 16‐week old male C57BL/6 mice were euthanized and first order mesenteric arteries were isolated for in vitro study. Endothelium‐dependent (acetylcholine) and ‐independent (sodium nitroprusside) vasodilator responses were evaluated with and without an acute EPO (50 mIU/mL) or EPOp (25 ng/mL) pretreatment. We found that acetylcholine, endothelium‐dependent, vasodilation was impaired in arteries pretreated with both EPO and EPOp as compared to controls. However, while the EPOp vasodilation was impaired by 60%, EPO pretreatment totally inhibited acetylcholine‐induced vasodilation. Importantly EPO, EPOp and control treated arteries were all able to vasodilate equally in response to sodium nitroprusside, an endothelium‐independent vasodilator. These findings suggest that acute activation of the homodimeric EPOR2 or the heterodimeric EPOR/βCR can both lead to an endothelial specific impairment of vasodilator function in resistance arteries. We will discuss the putative mechanism of these effects along with the physiologic significance.

Acute Reduction of Ambient Air Pressure Enhances Arterial Vasodilation in Murine Resistance Arteries Independently of Endothelial Factors

IntroductionEpidemiological studies have found populations living at higher elevations are at lower risk for acute myocardial infarction (MI) than those at lower elevations, even after adjustments for confounders. The mechanism for this observation is not known. Since atmospheric air pressure varies inversely with elevation, and air pressure exerts compressive force on vascular structures, reduced compressive force on arteries due to elevation‐related reductions in atmospheric pressure might alter arterial function. This could be a potential mechanism. We hypothesized that acute exposure to lower air pressure will dilate arteries and increase arterial vasodilation to reduce peripheral vascular resistance.MethodsSecond order mesenteric arteries (n=7) were isolated from C57BL/6 male mice (n=7, age 3.2±0.9 months) and perfused with Ca2+‐free saline solution to assess passive properties of the vessels in a pressure myograph. This system was placed in a custom‐designed hypobaric chamber and arterial function was studied ex‐vivo at acute exposure to three barometric pressure steps: 754 mmHg (room pressure or control), 714 mmHg (p1) and 674 mmHg (p2). Active vessel responses in the presence of L‐NAME, a nitric oxide inhibitor, and Meclofenamate, a cyclooxygenase inhibitor, were assessed with mechanical manipulation of perfusion pressure (4–140 mmHg) or flow rate (0–70 μL/min). Lumen diameter was measured using a micrometer, with manometers for measurement of pressure drop across the vessel.ResultsUnder static conditions, vessel diameters at p1 and p2 increased by 20.9±9.3% and 28.2±8.6% compared to baseline diameter at room pressure (p<0.01 vs. control for p1 and p2). Flow‐mediated vasodilation contributed little additional dilation. At a fixed flow rate of 70 μLmin−1, vessels reached maximum diameters of 108.6 ±13.6μm at p1 and 120.5±13.0 at p2 versus 106.0±4.0 μm for the control condition. This observed vasodilation was not diminished in the presence of L‐NAME and Meclofenamate. Vascular resistance was significantly reduced at p2 compared to control (2.14±0.60 mmHg*min/μL vs. 3.21±0.49 mmHg*min/μL, p < 0.05).ConclusionWe conclude that acute exposure to reduced barometric pressure increases artery diameter and decreases resistance across vessels ex‐vivo in an endothelium‐independent manner.

Long Lasting Microvascular Tone Alteration in Rat Offspring Exposed In Utero to Maternal Hyperglycaemia.

Epidemiologic studies have demonstrated that cardiovascular risk is not only determined by conventional risk factors in adulthood, but also by early life events which may reprogram vascular function. To evaluate the effect of maternal diabetes on fetal programming of vascular tone in offspring and its evolution during adulthood, we investigated vascular reactivity of third order mesenteric arteries from diabetic mother offspring (DMO) and control mother offspring (CMO) aged 3 and 18 months. In arteries isolated from DMO the relaxation induced by prostacyclin analogues was reduced in both 3- and 18-month old animals although endothelium (acetylcholine)-mediated relaxation was reduced in 18-month old DMO only. Endothelium-independent (sodium nitroprusside) relaxation was not affected. Pressure-induced myogenic tone, which controls local blood flow, was reduced in 18-month old CMO compared to 3-month old CMO. Interestingly, myogenic tone was maintained at a high level in 18-month old DMO even though agonist-induced vasoconstriction was not altered. These perturbations, in 18-months old DMO rats, were associated with an increased pMLC/MLC, pPKA/PKA ratio and an activated RhoA protein. Thus, we highlighted perturbations in the reactivity of resistance mesenteric arteries in DMO, at as early as 3 months of age, followed by the maintenance of high myogenic tone in older rats. These modifications are in favour of excessive vasoconstrictor tone. These results evidenced a fetal programming of vascular functions of resistance arteries in adult rats exposed in utero to maternal diabetes, which could explain a re-setting of vascular functions and, at least in part, the occurrence of hypertension later in life.

Estrogens are needed for the improvement in endothelium-mediated dilation induced by a chronic increase in blood flow in rat mesenteric arteries

Resistance arteries play a key role in the control of local blood flow. They undergo outward remodeling in response to a chronic increase in blood flow as seen in collateral artery growth in ischemic disorders. We have previously shown that mesenteric artery outward remodeling depends on the endothelial estrogen receptor alpha. As outward arterial remodeling is associated with improved endothelium-dependent dilation, we hypothesized that estrogens might also play a role in flow-mediated improvement of endothelium-dependent dilation.Local increase in blood flow in first order mesenteric arteries was obtained after ligation of adjacent arteries in three-month old ovariectomized female rats treated with 17-beta-estradiol (OVX+E2) or vehicle (OVX).After 2weeks, diameter was equivalent in high flow (HF) than in normal flow (NF) arteries with a greater wall to lumen ratio in HF vessels in OVX rats. Acetylcholine-mediated relaxation was lower in HF than in NF vessels. eNOS and caveolin-1 expression level was equivalent in HF and NF arteries.By contrast, arterial diameter was 30% greater in HF than in NF arteries and the wall to lumen ratio was not changed in OVX+E2 rats. Acetylcholine-mediated relaxation was higher in HF than in NF arteries. The expression level of eNOS was higher and that of caveolin-1 was lower in HF than in NF arteries.Acetylcholine (NO-dependent)-mediated relaxation was partly inhibited by the NO-synthesis blocker L-NAME in OVX rats whereas L-NAME blocked totally the relaxation in OVX+E2 rats. Endothelium-independent relaxation (sodium nitroprusside) was equivalent in OXV and OVX+E2 rats. Similarly, serotonin- and phenylephrine-mediated contractions were higher in HF than in NF arteries in both OVX and OVX+E2 rats in association with high ratio of phosphorylated ERK1/2 to ERK1/2.Thus, we demonstrated the essential role of endogenous E2 in flow-mediated improvement of endothelium (NO)-mediated dilatation in rat mesenteric arteries.