Pressure Myography Research Articles

32 Premature vascular ageing in hypertension: role of aldosterone and nadph oxidases

In hypertension, the vasculature undergoes premature ageing. The role of aldosterone and NADPH oxidases (Nox’s) in the molecular mechanisms of age-associated vascular damage is unclear. We hypothesise that premature ageing in hypertension is due to increased aldosterone-induced Nox activation. We assessed vascular ageing in resistance arteries and vascular smooth muscle cells (VSMCs) from adult WKY (18 weeks), aged WKY (52 weeks) and adult stroke-prone spontaneously hypertensive (SHRSP) rats. Blood pressure (BP) was measured by tail-cuff. Vascular structure and function were analysed by pressure and wire myography. Gene and protein expression were assessed by qPCR and immunoblotting. We observed elevated BP, endothelial dysfunction and hypercontractility in vessels from SHRSP (vs. WKY). Vascular contraction in aged WKY rats was similar to SHRSP rats. Increased stiffness was observed in mesenteric arteries from aged WKY and SHRSP rats (vs. WKY). Nox2, NoxA1 and NoxO1 mRNA expression was increased (p

Implications of α v β 3 Integrin Signaling in the Regulation of Ca 2+ Waves and Myogenic Tone in Cerebral Arteries

The myogenic response is central to blood flow regulation in the brain. Its induction is tied to elevated cytosolic [Ca(2+)], a response primarily driven by voltage-gated Ca(2+) channels and secondarily by Ca(2+) wave production. Although the signaling events leading to the former are well studied, those driving Ca(2+) waves remain uncertain. We postulated that αvβ3 integrin signaling is integral to the generation of pressure-induced Ca(2+) waves and cerebral arterial tone. This hypothesis was tested in rat cerebral arteries using the synergistic strengths of pressure myography, rapid Ca(2+) imaging, and Western blot analysis. GRGDSP, a peptide that preferentially blocks αvβ3 integrin, attenuated myogenic tone, indicating the modest role for sarcoplasmic reticulum Ca(2+) release in myogenic tone generation. The RGD peptide was subsequently shown to impair Ca(2+) wave generation and myosin light chain 20 (MLC20) phosphorylation, the latter of which was attributed to the modulation of MLC kinase and MLC phosphatase via MYPT1-T855 phosphorylation. Subsequent experiments revealed that elevated pressure enhanced phospholipase Cγ1 phosphorylation in an RGD-dependent manner and that phospholipase C inhibition attenuated Ca(2+) wave generation. Direct inhibition of inositol 1, 4, 5-triphosphate receptors also impaired Ca(2+) wave generation, myogenic tone, and MLC20 phosphorylation, partly through the T-855 phosphorylation site of MYPT1. Our investigation reveals a hitherto unknown role for αvβ3 integrin as a cerebral arterial pressure sensor. The membrane receptor facilitates Ca(2+) wave generation through a signaling cascade, involving phospholipase Cγ1, inositol 1,3,4 triphosphate production, and inositol 1, 4, 5-triphosphate receptor activation. These discrete asynchronous Ca(2+) events facilitate MLC20 phosphorylation and, in part, myogenic tone by influencing both MLC kinase and MLC phosphatase activity.

Unitary TRPV3 channel Ca2+ influx events elicit endothelium-dependent dilation of cerebral parenchymal arterioles.

Cerebral parenchymal arterioles (PA) regulate blood flow between pial arteries on the surface of the brain and the deeper microcirculation. Regulation of PA contractility differs from that of pial arteries and is not completely understood. Here, we investigated the hypothesis that the Ca(2+) permeable vanilloid transient receptor potential (TRPV) channel TRPV3 can mediate endothelium-dependent dilation of cerebral PA. Using total internal reflection fluorescence microscopy (TIRFM), we found that carvacrol, a monoterpenoid compound derived from oregano, increased the frequency of unitary Ca(2+) influx events through TRPV3 channels (TRPV3 sparklets) in endothelial cells from pial arteries and PAs. Carvacrol-induced TRPV3 sparklets were inhibited by the selective TRPV3 blocker isopentenyl pyrophosphate (IPP). TRPV3 sparklets have a greater unitary amplitude (ΔF/F0 = 0.20) than previously characterized TRPV4 (ΔF/F0 = 0.06) or TRPA1 (ΔF/F0 = 0.13) sparklets, suggesting that TRPV3-mediated Ca(2+) influx could have a robust influence on cerebrovascular tone. In pressure myography experiments, carvacrol caused dilation of cerebral PA that was blocked by IPP. Carvacrol-induced dilation was nearly abolished by removal of the endothelium and block of intermediate (IK) and small-conductance Ca(2+)-activated K(+) (SK) channels. Together, these data suggest that TRPV3 sparklets cause dilation of cerebral parenchymal arterioles by activating IK and SK channels in the endothelium.

Impaired endothelial calcium signaling is responsible for the defective dilation of mesenteric resistance arteries from db/db mice to acetylcholine

We aimed at assessing the role of endothelial cell calcium for the endothelial dysfunction of mesenteric resistance arteries of db/db mice (a model of type 2 diabetes) and determine whether treatment with sulfaphenazole, improves endothelial calcium signaling and function. Pressure myography was used to study acetylcholine (ACh) -induced vasodilation. Intracellular calcium ([Ca2+]i) transients was measured by confocal laser scanning microscopy and smooth muscle membrane potential with sharp microelectrodes. The impaired dilation to ACh observed in mesenteric resistance arteries from db/db mice was improved by treatment of the mice with sulfaphenazole for 8 weeks. The impaired dilation to ACh was associated with decreased endothelial [Ca2+]i and smooth muscle hyperpolarization. Sulfaphenazole applied in vitro improved endothelial mediated dilation of arteries from db/db mice both in the absence and the presence of inhibitors of nitric oxide and cyclooxygenase. Sulfaphenazole also increased the percentage of endothelial cells with ACh induced increases of [Ca2+]i. The study shows that impaired endothelial [Ca2+]i control can explain the reduced endothelial function in arteries from diabetic mice and that sulfaphenazole treatment improves endothelial [Ca2+]i responses to ACh and consequently endothelium-dependent vasodilation. These observations provide mechanistic insight into endothelial dysfunction in diabetes.

Sphingosine-1-Phosphate Signaling Regulates Myogenic Responsiveness in Human Resistance Arteries.

We recently identified sphingosine-1-phosphate (S1P) signaling and the cystic fibrosis transmembrane conductance regulator (CFTR) as prominent regulators of myogenic responsiveness in rodent resistance arteries. However, since rodent models frequently exhibit limitations with respect to human applicability, translation is necessary to validate the relevance of this signaling network for clinical application. We therefore investigated the significance of these regulatory elements in human mesenteric and skeletal muscle resistance arteries. Mesenteric and skeletal muscle resistance arteries were isolated from patient tissue specimens collected during colonic or cardiac bypass surgery. Pressure myography assessments confirmed endothelial integrity, as well as stable phenylephrine and myogenic responses. Both human mesenteric and skeletal muscle resistance arteries (i) express critical S1P signaling elements, (ii) constrict in response to S1P and (iii) lose myogenic responsiveness following S1P receptor antagonism (JTE013). However, while human mesenteric arteries express CFTR, human skeletal muscle resistance arteries do not express detectable levels of CFTR protein. Consequently, modulating CFTR activity enhances myogenic responsiveness only in human mesenteric resistance arteries. We conclude that human mesenteric and skeletal muscle resistance arteries are a reliable and consistent model for translational studies. We demonstrate that the core elements of an S1P-dependent signaling network translate to human mesenteric resistance arteries. Clear species and vascular bed variations are evident, reinforcing the critical need for further translational study.

Abstract P062: Long-term Exposure to Endothelin-1 Overexpression Increases Blood Pressure and Causes Small Artery Injury

Background: The mechanisms of blood pressure (BP) regulation by endothelin (ET)-1 produced by endothelial cells are complex and remain unclear. Recently, we developed a transgenic mouse with tamoxifen-inducible endothelium-restricted human ET-1 overexpression (ieET-1) using Cre/loxP technology. ieET-1 mice exhibited BP rise after three weeks of induction in an ET type A receptor-dependent manner, in absence of vascular and kidney injury. It is unknown whether long-term exposure to ET-1 overexpression results in elevated BP elevation and vascular injury. Methods: Nine to 12-week old male ieET-1 mice and control ieCre mice expressing a tamoxifen-inducible Cre recombinase (CreERT2) under the control of EC-specific Tie2 promoter, were treated with tamoxifen (1 mg/kg/day, s.c.) for 5 days and studied 3 months later. Metabolic cages were used to collect 24-hour urine for sodium, potassium and protein measurements. Renal artery flow (RAF) was assessed by ultrasonography. BP was determined by telemetry, plasma aldosterone by ELISA, and small mesenteric artery (MA) endothelial function and vascular remodeling by pressurized myography. Results: Systolic BP was increased in ieET-1 compared with ieCre mice (144±5 vs 117±3 mmHg, P<0.001). RAF was decreased in ieET-1 compared with control (1.9±0.2 vs 3.0±0.3 mL/min, P<0.01). The excretion of urinary sodium, potassium and protein was similar in both groups. Plasma aldosterone levels were increased in ieET-1 compared with ieCre mice (1.99±0.20 vs 1.29±0.12 ng/mL, P<0.05). Endothelium-dependent relaxation responses to acetylcholine were impaired in ieET-1 compared to ieCre mice (36.3±4.7 vs 71.4±9.7%, P<0.01), whereas endothelium-independent relaxation responses to sodium nitroprusside were unchanged. MA media/lumen and media cross-sectional area were similar in both groups, but stiffness was increased in ieET-1 compared to ieCre mice, as indicated by leftward displacement of the stress-strain curves (strain at 140mmHg: 0.61±0.04 vs 0.71±0.02, P<0.05). Conclusions: The results demonstrate that long-term exposure to endothelial ET-1 overexpression caused sustained BP rise and small artery stiffening.

Abstract P015: Bilateral Common Carotid Artery Stenosis in Hypertensive Rats Impairs Dilation in Penetrating Arterioles and Posterior Communicating Arteries

Hypertension and chronic cerebral hypoperfusion (CCH) are leading risk factors for cognitive impairment. We induced hypoperfusion in stroke prone spontaneously hypertensive rats (SHRSP) by bilateral carotid artery stenosis (BCAS) to create a novel, physiologically relevant model of cognitive impairment. We hypothesized that BCAS in SHRSP would impair endothelium dependent dilation and lead to outward remodeling of the penetrating arterioles (PAs) and posterior cerebral arteries (PCAs). The PAs are critical for maintaining parenchymal perfusion and controlling blood flow to the neurovascular unit, and the PCAs are integral in regulating the severity of CCH. Both artery types were studied by pressure myography and data are shown as mean±SEM, SHAM vs BCAS (an n =4 to 12 in each group) with p<0.05. Short-term memory, evaluated by novel object recognition testing, was impaired 8 weeks after BCAS (novel exploration quotient: 0.60±0.1 vs 0.45±0.1). Endothelium-dependent dilation was assessed with carbachol (data shown as % dilation at 10 -6 M). BCAS impaired endothelial function in PAs, as evidenced by the abolition of the dilation (14±3.3% vs -10±4.7%) but did not significantly alter sodium nitroprusside induced dilation (60 ± 9.7% vs. 49 ± 6.4%). Inhibition of nitric oxide and prostacyclin production with L-NAME (100 μM) and indomethacin (10 μM), respectively, did not change dilation in either group. This suggests that endothelium-derived hyperpolarizing factor plays a role in PA dilation in SHRSP. PAs from BCAS rats had a reduced wall thickness (at 60 mmHg: 7±0.5 vs 5±0.6 μm), and wall-to-lumen ratio (at 60 mmHg: 0.10±0.002 vs 0.07±0.0001). BCAS increased wall stress in PAs (at 60 mmHg: 306±30.2 vs 427±48.3 dynes/cm 2 ). Endothelial dysfunction also was evident in the PCAs after BCAS (50 ± 13.0% vs 3 ± 14.9%) but the pattern of remodeling in these larger arteries was different. We did not observe a change in the wall thickness or wall-to-lumen ratio in PCAs after BCAS, but the lumen diameter was increased (at 80 mmHg: 105±7.7 vs 116±5.7 μm). These data suggest that endothelial dysfunction in the PAs and PCAs may worsen the severity of cerebral hypoperfusion and cognitive decline in SHRSP. The thinner walls in the PAs may also increase the risk of hemorrhage in the BCAS rats.

Abstract P601: Matrix Metalloproteinase 2 Plays An Important Role In Angiotensin Ii-induced Vascular Injury Mediated In Part Through Epidermal Growth Factor Receptor Activation In Vascular Smooth Muscle Cells

Background: Matrix metalloproteinase 2 (MMP2) is involved in cardiovascular disease. Whether MMP2 plays a role in hypertension and vascular damage is unknown. We hypothesized that Mmp2 knockout will prevent angiotensin (Ang) II-induced blood pressure (BP) rise and vascular injury. Methods: Ten to 12-week-old male Mmp2 knockout (Mmp2-/-) and wild-type (WT) mice were infused with Ang II (1000 ng/kg/min, SC) for 14 days. Systolic BP was measured by telemetry, mesenteric arteries (MA) endothelial function and vascular remodeling by pressurized myography. In aortic wall or perivascular fat (PVAT), reactive oxygen species (ROS) generation was determined using dihydroethidium staining, and vascular cell adhesion protein 1 (VCAM-1), monocyte chemotactic protein-1 (MCP-1) expression and monocyte/macrophage infiltration by immunofluorescence. Spleen T cells and monocyte profile were assessed by flow cytometry. Vascular smooth muscle cells (VSMCs) were isolated from MA of WT and Mmp2 knockout mice, stimulated 5 min with 100 nM Ang II and epidermal growth factor receptor (EGFR) phosphorylation measured by Western-Blotting. Results: Ang II increased Systolic BP (172±7 vs 122±3, P<0.01), decreased MA vasodilatory responses to acetylcholine (33±5% vs 83±3%, P<0.01) and increases MA media-to-lumen ratio (5±0% vs 3±0%, P<0.01), media cross-sectional area (7224±467 vs 5345±336 μm2, P<0.05), and stiffness (P<0.01), as shown by a leftward shift of the stress/strain relationship, in WT. Furthermore, Ang II enhanced aortic ROS generation (73±11 vs 6±1 RFU/μm2, P<0.01), aortic VCAM-1 (17±3 vs 5±3 RFU/μm2, P<0.01) and MCP-1 expression (71±14 vs 11±3 RFU/μm2, P<0.01) and PVAT monocyte/macrophage infiltration (32±5 vs 4±0 RFU/μm2, P<0.05), and spleen activated CD4+CD69+ and CD8+CD69+ T cells and pro-inflammatory Ly-6Chi monocytes (17±2 vs 10±1%, 11±1 vs 5±1% and 53±6 vs 25±2%, respectively, P<0.05) in WT. Ang II increased EGFR phosphorylation in VSMCs in vitro (1.91±0.19 vs 1.0±0.0%, P<0.05). Mmp2 knockout prevented or reduced all of the above except BP elevation (P<0.05). Conclusion: MMP2 plays an important role in Ang II-induced vascular injury, which could be mediated at least in part through EFGR activation in VSMCs.

Abstract P617: Angiotensin II-induced Hypertension And Vascular Injury Is Mediated By Gamma/delta T Cells

Objective: Both innate antigen presenting cells and the adaptive immune system have been shown to play a role in the development of hypertension. Nevertheless, the T cell subset involved in the pathophysiology of hypertension remains unclear. There is a small subset of “innate-like” T cells expressing gamma/delta T cell receptor (TCR) rather than the alpha/beta TCR that could play a role in bridging between the innate and adaptive immune systems. However, it is unknown whether gamma/delta T cells contribute to development of hypertension. Method/Results: Thirteen to 15 week-old male C57BL/6 wild-type and Tcrd-/- mice, which are devoid of gamma/delta T cells, were infused or not with angiotensin (Ang) II (490 ng/kg/min, SC) for 7 or 14 days (n=4-9). Telemetric blood pressure, mesenteric artery endothelial function and vascular remodeling by pressurized myography and spleen T cell profile by flow cytometry were evaluated. Fourteen days of Ang II increased systolic blood pressure (167±4 vs 125±2 mmHg, P≤0.01) in wild-type compared to control mice. The frequency of gamma/delta T cells (6±1% vs 3±1%, P≤0.05) and activated (CD69+) gamma/delta T cells (11±1% vs 7±1%) was increased after 7 days of Ang II, and 7 days later were respectively unchanged or further increased (24±2% vs 10±1%) in wild-type compared to control mice. Ang II decreased mesenteric artery relaxation responses to acetylcholine (51±5% vs 88±3%, P≤0.01) and increased media/lumen (5±1 vs 3±0%, P≤0.01) in wild-type mice compare to controls. No gamma/delta T cells were detected in Tcrd-/- treated or not with Ang II. All the above Ang II effects were abrogated in Tcrd-/- mice. Conclusion: These data suggest that gamma/delta T cells mediate Ang II-induced blood pressure rise and vascular injury. Gamma/delta T cells could be key immune cells bridging innate and adaptive immune responses during the development of hypertension.

Abstract 107: Cytochrome B5 Reductase 3 Sensitizes Soluble Guanylate Cyclase to Nitric Oxide in Vascular Smooth Muscle

The inability nitric oxide (NO) to stimulate soluble guanylate cyclase (sGC) has been linked to numerous cardiovascular diseases (CVD) including hypertension. While several studies have defined the importance of sGC expression in the cardiovascular system, the basic mechanisms that regulate sGC activity remain incompletely understood. Here, we report for the first time that sGC heme iron redox state, which is essential for NO-induced sGC activation, is regulated by cytochrome B5 reductase 3 (CyB5R3). Genetic knockdown and pharmacological inhibition of CyB5R3 in primary rat vascular smooth muscle cells resulted in a 60% loss in cGMP production. Conversely, the sGC activator Bay 58-2667, which activates oxidized or heme free sGC, reversed these effects. Consistent with our cell culture work, purified protein studies demonstrate that CyB5R3 can directly reduce oxidized sGC heme iron and sensitize sGC to NO. To test the functional importance of Cyb5R3 activity, we cultured mouse thoracodorsal arteries with a pharmacological inhibitor of Cyb5R3 (ZINC 747) and performed vascular reactivity studies using pressure myography. Arteries treated with ZINC 747 showed decreased responsiveness the NO donor DETA-NONOate but increase sensitivity to Bay 58-2667. We then treated mice with 10mg/kg/day of ZINC 747 using osmotic mini pumps, which caused an increase in mean arterial blood pressure (107.5±3.4 vs 131±13.16) measured via radio telemetry. Lastly, translational studies reveal that the CyB5R3 T116S polymorphism with allele frequency 0.23 only in African Americans is unable to reduce sGC and correlates with increased blood pressure. Considering the defining role of sGC in NO signaling and the fact that the oxidation state of sGC may predict responses to NO therapies and new classes of sGC activator medications, we anticipate that these studies may significantly impact our understanding of biology, precision therapeutics (right drug for the right patient) and pharmacogenetics (T117S SNP based drug selection).

Abstract P257: High Fat Diet Induces Metabolic Syndrome-Like Phenotype Associated with Adverse Micro- and Macro-Vascular Remodeling

Metabolic diseases such as type 2 diabetes (T2DM), hypertension, and metabolic syndrome (MetS) have been associated with vascular disease, and we have previously demonstrated vascular-bed-specific remodeling in both mouse and porcine models of T2DM and MetS. The aim of this study was to determine whether high-fat diet would induce MetS-associated adverse micro- and macro-vascular remodeling and mechanics in mice. Three week old male C57BL/6J mouse siblings were randomized to receive either a normal low-fat (LFD: 10% fat) or high-fat (HFD: 60% fat) diet for 20 weeks (n=7-10 per group). HFD induced a MetS-like phenotype characterized by increased body weight (LFD: 28.6±0.7 vs. HFD: 43.4±0.8 g, p <0.0001), increased mean arterial pressure (LFD: 65±3 vs. HFD: 91±2 mmHg, p <0.0001), increased plasma insulin (LFD: 106±39 vs. HFD: 368±54 pg/mL, p <0.001), and transient increases in fasting blood glucose. Passive pressure myography of septal coronary resistance microvessels (CRMs) revealed reduced internal (LFD: 151±11 vs. HFD: 113±7 μm at 125 mmHg, p <0.05) and external diameters, increased wall/lumen ratio (LFD: 5.5±0.6 vs. HFD: 7.8±0.5 at 125 mmHg, p <0.01) and reduced incremental modulus of elasticity (LFD: 7.9x10 6 ±1.7x10 6 vs. HFD: 4.5x10 6 ±0.6x10 6 dynes/cm 2 at 125 mmHg, p <0.01) in mice fed HFD. Adverse CRM remodeling was associated with reduced coronary flow at baseline and under hyperemic conditions, which reduced coronary flow reserve (LFD: 7.3±0.5 vs. HFD: 5.5±0.5, p <0.05). Aortic pulse wave velocity was increased (LFD: 0.31±0.02 vs. HFD: 0.36±0.01 cm/ms, p <0.05) and significantly correlated with the increased blood pressure (r=0.67, p<0.01). These data demonstrate that 20 weeks of a high-fat diet induces an early MetS-like pathophysiological state that is associated with vascular-bed-specific remodeling and alterations in vascular biomechanics. Furthermore, the presence of adverse vascular remodeling in the presence of an early MetS-like phenotype, but not overt MetS (i.e. in the presence of sustained elevation in fasting blood glucose), may suggest the presence of underlying sub-clinical disease during the early progression of metabolic syndrome.

Abstract 067: Endothelial S1P/S1P1 Signaling is Critical Regulator of Blood Flow and Pressure

Background and objectives: Sphingosine-1-phosphate (S1P) is emerging as a pivotal signaling molecule within the vasculature. S1P has been known as a potent endothelial nitric oxide synthase activator through G-protein coupled receptor S1P1 and less abundant S1P3 on endothelial cells (EC). Although it has been recently shown that S1P/S1P1 signaling mediates EC alignment in response to flow, the role of S1P1 signaling in the pathogenesis of hypertension remains unknown. FTY720, an S1P1 functional antagonist, recently approved by the FDA to treat autoimmune conditions, induces a modest and transient decrease in heart rate in both animals and humans, suggesting that drugs targeting sphingolipid signaling affect cardiovascular functions in vivo. Thus, the aim of this study is to investigate whether S1P-S1P1 signaling pathway is crucial in blood flow and pressure regulation. Methods: The BP was evaluated in conscious mice with tail cuff system, whereas the vascular reactivity was examined with pressure myograph system in mesenteric arteries isolated from age matched WT and EC-S1P1-/- mice. Results. Here we report for the first time that S1P1-mediated signaling protects from hypertension. Administration of SEW 2871, an agonist of S1P1, to angiotensin-II-induced hypertensive mice lowered blood pressure to normotensive levels (110.7±3.1 vs. 160.7±2.4 mmHg, SEW 2871 vs. vehicle respectively). Pharmacological inhibition of S1P1 with W146 (100 nM and 1 μM) markedly reduced flow-induced vasodilation (Emax 25.2±3.8 and 2.5±2.3 vs. 48.6±4.9 μm, W146 100 nM and 1μM respectively vs. WT). These data were corroborated by using a genetic approach: mice EC-S1P1-/- were hypertensive (124.7±1.9 vs. 109±1.7 mmHg, EC-S1P1-/- vs. S1P1f/f mice) and showed a reduced response to flow-induced vasodilation (Emax 15.2±3.0 vs. 53.8±5.0 μm, EC-S1P1-/- vs. S1P1f/f mice mesenteric artery). Conclusion: Our study identifies S1P-S1P1 signaling as a new regulatory pathway of blood flow and pressure homeostasis, providing a novel therapeutic target for the treatment of hypertension.

CaV3.1 T-Type Ca2+ Channels Contribute to Myogenic Signaling in Rat Retinal Arterioles.

Although L-type Ca2+ channels are known to play a key role in the myogenic reactivity of retinal arterial vessels, the involvement of other types of voltage-gated Ca2+ channels in this process remains unknown. In the present study we have investigated the contribution of T-type Ca2+ channels to myogenic signaling in arterioles of the rat retinal microcirculation. Confocal immunolabeling of whole-mount preparations was used to investigate the localization of CaV3.1-3 channels in retinal arteriolar smooth muscle cells. T-type currents and the contribution of T-type channels to myogenic signaling were assessed by whole-cell patch-clamp recording and pressure myography of isolated retinal arteriole segments. Strong immunolabeling for CaV3.1 was observed on the plasma membrane of retinal arteriolar smooth muscle cells. In contrast, no expression of CaV3.2 or CaV3.3 could be detected in retinal arterioles, although these channels were present on glial cell end-feet surrounding the vessels and retinal ganglion cells, respectively. TTA-A2-sensitive T-type currents were recorded in retinal arteriolar myocytes with biophysical properties distinct from those of the L-type currents present in these cells. Inhibition of T-type channels using TTA-A2 or ML-218 dilated isolated, myogenically active, retinal arterioles. CaV3.1 T-type Ca2+ channels are functionally expressed on arteriolar smooth muscle cells of retinal arterioles and play an important role in myogenic signaling in these vessels. The work has important implications concerning our understanding of the mechanisms controlling blood flow autoregulation in the retina and its disruption during ocular disease.

Ghrelin restores nitric oxide availability in resistance circulation of essential hypertensive patients: role of NAD(P)H oxidase.

We assessed whether acute intra-arterial infusion of exogenous ghrelin can improve endothelial dysfunction by restoring nitric oxide (NO) availability in the forearm microcirculation of essential hypertensive patients. The effect of ghrelin on endothelial dysfunction (pressurized myograph), vascular oxidative stress generation (fluorescent dihydroethidium), and phosphorylation of p47phox (western blot), an index of NAD(P)H oxidase activation, in isolated small arteries taken from essential hypertensive patients (subcutaneous biopsy) were also investigated. In 18 normotensive control subjects and 18 essential hypertensive patients, we studied the forearm blood flow (strain-gauge plethysmography) response to intra-arterial acetylcholine, repeated under NO synthase inhibitor N(G)-monomethyl-l-arginine (l-NMMA) or the antioxidant ascorbic acid. The protocol was repeated at the end of exogenous ghrelin intra-arterial infusion. In hypertensive patients, ghrelin normalized the blunted response to acetylcholine, restored the inhibiting effect of l-NMMA and abrogated the potentiating effect of ascorbic acid on acetylcholine. In controls, ghrelin failed to modify these vascular responses. In hypertensive patients, ghrelin decreased venous levels of malondialdehyde, lipoperoxide, and interleukin-6, and concomitantly increased endogenous antioxidant capacity. Small vessels from hypertensive patients showed an enhanced intravascular oxidative stress, which was strongly and similarly decreased by incubation with ghrelin, the NAD(P)H oxidase inhibitor gp91 ds-tat, or both. Ghrelin also normalized the overexpression of p47 phosphorylation and restored the NO availability in small vessels from hypertensive patients. Exogenous ghrelin increases endothelial dysfunction by restoring NO availability in the forearm microcirculation of essential hypertensive patients, an effect ascribable to an antioxidant effect via inhibition of NAD(P)H oxidase activation.

Abstract 179: Targeted Inhibition of Plasminogen Activator Inhibitor-1 Attenuates Weight Gain and Prevents Vascular Dysfunction Following a High Fat Diet

Introduction: Elevated plasminogen activator inhibitor-1 (PAI-1) is associated with obesity, but there is controversy whether PAI-1 causes or is a consequence of obesity. We sought to determine whether targeted PAI-1 inhibition with a novel small molecule antagonist (TM5441) alters the development of obesity and/or obesity-induced vascular dysfunction in a diet-induced obesity model. Methods and Results: C57BL/6J mice were fed control, high fat diet (HFD), or high fat diet with TM5441 (HFD+TM5441) for 12 weeks. The HFD had marked weight gain (77±5%) as compared with control (32±2%). TM5441 significantly attenuated weight gain (49±8%, p=0.0075, Figure). HFD-induced hepatic triglyceride accumulation was attenuated by TM5441 (116±31 vs. 76±35 mg trig/g liver, p=0.03). Energy expenditure was reduced in the HFD compared to control (11.1±0.4 vs. 12.9±0.4 kcal/h/kg, p=0.005). However, HFD+TM5441 maintained a level of energy expenditure that was similar to control (13.2±0.6 kcal/h/kg, p=NS). The HFD group demonstrated higher systolic and diastolic blood pressure (141±3; 112±3 mm Hg) compared with control (122±7, 94±8; P<0.05 for both), while administration of TM5441 prevented diet-associated increase (120±6; 93±7 mm Hg, p=NS compared to control) at week 12. Pressure myography of mesenteric arteries in the HFD showed a significant rightward shift in the constrictor response to phenylephrine as compared to control (EC50: 14.5uM vs. 25.1uM, p=0.002). The HFD+TM5441 was similar to control (p=NS). Conclusions: Inhibition of PAI-1 with TM5441 attenuates weight gain, enhances energy expenditure, and prevents obesity-related vascular dysfunction in a murine model of obesity.

Vasopeptidase Inhibition Corrects the Structure and Function of the Small Arteries in Experimental Renal Insufficiency

Background: We studied whether vasopeptidase inhibition corrects the structure and function of the small arteries in experimental chronic renal insufficiency (CRI). Methods: After 5/6 nephrectomy (NX) surgery was performed on rats, there was a 14-week follow-up, allowing CRI to become established. Omapatrilat (40 mg/kg/day in chow) was then given for 8 weeks, and the small mesenteric arterial rings were investigated in vitro using wire and pressure myographs. Results: Plasma and ventricular B-type natriuretic peptide (BNP) concentrations were increased 2- to 2.7-fold, while systolic blood pressure (BP) increased by 32 mm Hg after NX. Omapatrilat treatment normalized the BNP and reduced the BP by 45 mm Hg in the NX rats. Endothelium-dependent vasorelaxation was impaired but the response to acetylcholine was normalized after omapatrilat treatment. Vasorelaxations induced by nitroprusside, isoprenaline and levcromakalim were enhanced after omapatrilat, and the responses were even more pronounced than in untreated sham-operated rats. Arterial wall thickness and wall-to-lumen ratio were increased after NX, whereas omapatrilat normalized these structural features and improved the strain-stress relationship in the small arteries; this suggests improved arterial elastic properties. Conclusion: Omapatrilat treatment reduced BP, normalized volume overload, improved vasorelaxation and corrected the dimensions and passive elastic properties of the small arteries in the NX rats. Therefore, we consider vasopeptidase inhibition to be an effective treatment for CRI-induced changes in the small arteries.

Assessing Myogenic Response and Vasoactivity In Resistance Mesenteric Arteries Using Pressure Myography.

Small resistance arteries constrict and dilate respectively in response to increased or decreased intraluminal pressure; this phenomenon known as myogenic response is a key regulator of local blood flow. In isobaric conditions small resistance arteries develop sustained constriction known as myogenic tone (MT), which is a major determinant of systemic vascular resistance (SVR). Hence, ex vivo pressurized preparations of small resistance arteries are major tools to study microvascular function in near-physiological states. To achieve this, a freshly isolated intact segment of a small resistance artery (diameter ~260 μm) is mounted onto two small glass cannulas and pressurized. These arterial preparations retain most in vivo characteristics and permit assessment of vascular tone in real-time. Here we provide a detailed protocol for assessing vasoactivity in pressurized small resistance mesenteric arteries from rats; these arteries develop sustained vasoconstriction - approximately 25% of maximal diameter - when pressurized at 70 mmHg. These arterial preparations may be used to study the effect of investigational compounds on relationship between intra-arterial pressure and vasoactivity and determine changes in microvascular function in animal models of various diseases.

Assessing Myogenic Response and Vasoactivity In Resistance Mesenteric Arteries Using Pressure Myography

Assessing Myogenic Response and Vasoactivity In Resistance Mesenteric Arteries Using Pressure Myography

The angiotensin receptor blocker losartan reduces coronary arteriole remodeling in type 2 diabetic mice

Cardiovascular complications are a leading cause of morbidity and mortality in type 2 diabetes mellitus (T2DM) and are associated with alterations of blood vessel structure and function. Although endothelial dysfunction and aortic stiffness have been documented, little is known about the effects of T2DM on coronary microvascular structural remodeling. The renin–angiotensin–aldosterone system plays an important role in large artery stiffness and mesenteric vessel remodeling in hypertension and T2DM. The goal of this study was to determine whether the blockade of AT1R signaling dictates vascular smooth muscle growth that partially underlies coronary arteriole remodeling in T2DM. Control and db/db mice were given AT1R blocker losartan via drinking water for 4weeks. Using pressure myography, we found that coronary arterioles from 16-week db/db mice undergo inward hypertrophic remodeling due to increased wall thickness and wall-to-lumen ratio with a decreased lumen diameter. This remodeling was accompanied by decreased elastic modulus (decreased stiffness). Losartan treatment decreased wall thickness, wall-to-lumen ratio, and coronary arteriole cell number in db/db mice. Losartan treatment did not affect incremental elastic modulus. However, losartan improved coronary flow reserve. Our data suggest that Ang II–AT1R signaling mediates, at least in part, coronary arteriole inward hypertrophic remodeling in T2DM without affecting vascular mechanics, further suggesting that targeting the coronary microvasculature in T2DM may help reduce cardiac ischemic events.

Differential effects of relaxin deficiency on vascular aging in arteries of male mice.

Exogenous treatment with the naturally occurring peptide relaxin increases arterial compliance and reduces vascular stiffness. In contrast, relaxin deficiency reduces the passive compliance of small renal arteries through geometric and compositional vascular remodeling. The role of endogenous relaxin on passive mechanical wall properties in other vascular beds is unknown. Importantly, no studies have investigated the effects of aging in arteries of relaxin-deficient mice. Therefore, we tested the hypothesis that mesenteric and femoral arteries stiffen with aging, and this is exacerbated with relaxin deficiency. Male wild-type (Rln (+/+)) and relaxin knockout (Rln (-/-)) mice were aged to 3, 6, 12, 18, and 23 months. Passive mechanical wall properties were assessed by pressure myography. In both genotypes, there was a significant increase in circumferential stiffening in mesenteric arteries with aging, whereas in the femoral artery, aging reduced volume compliance. This was associated with a reduced ability of the artery to lengthen with aging. The predominant phenotype observed in Rln (-/-) mice was reduced volume compliance in young mice in both mesenteric and femoral arteries. In summary, aging induces circumferential stiffening in mesenteric arteries and axial stiffening in femoral arteries. Passive mechanical wall properties of Rln (-/-) mouse arteries predominantly differ at younger ages compared with Rln (+/+) mice, suggesting that a lack of endogenous relaxin only has a minor effect on vascular aging.