Responses Of Resistance Arteries Research Articles

PTPRG is an ischemia risk locus essential for HCO3--dependent regulation of endothelial function and tissue perfusion.

Acid-base conditions modify artery tone and tissue perfusion but the involved vascular-sensing mechanisms and disease consequences remain unclear. We experimentally investigated transgenic mice and performed genetic studies in a UK-based human cohort. We show that endothelial cells express the putative HCO3--sensor receptor-type tyrosine-protein phosphatase RPTPγ, which enhances endothelial intracellular Ca2+-responses in resistance arteries and facilitates endothelium-dependent vasorelaxation only when CO2/HCO3- is present. Consistent with waning RPTPγ-dependent vasorelaxation at low [HCO3-], RPTPγ limits increases in cerebral perfusion during neuronal activity and augments decreases in cerebral perfusion during hyperventilation. RPTPγ does not influence resting blood pressure but amplifies hyperventilation-induced blood pressure elevations. Loss-of-function variants in PTPRG, encoding RPTPγ, are associated with increased risk of cerebral infarction, heart attack, and reduced cardiac ejection fraction. We conclude that PTPRG is an ischemia susceptibility locus; and RPTPγ-dependent sensing of HCO3- adjusts endothelium-mediated vasorelaxation, microvascular perfusion, and blood pressure during acid-base disturbances and altered tissue metabolism.

Abstract P139: Cytochrome B5 Reductase 3 Biases Activation of Soluble Guanylyl Cyclase in Resistance Arteries

Soluble guanylyl cyclase (sGC) heme oxidation (Fe 3+ ) leading to apo-sGC formation diminishes the ability of nitric oxide (NO) to bind sGC and generate intracellular cGMP levels needed for downstream protein kinase G activation and vessel relaxation. While several studies suggest that pathological oxidative stress drives sGC heme oxidation and apo-sGC formation, we report for the first time that resistance arteries, but not conduit arteries, contain a previously unrecognized pool of heme oxidized and/or apo-sGC in non-stressed conditions that can be activated independent of NO. Specifically, our vascular reactivity studies indicate that systemic resistance and pulmonary arteries are responsive to NO-independent sGC activator BAY 58-2667 at 2-3 log-fold lower concentrations than that of conduit aorta and renal arteries (n=4-5/ vascular bed). Further, mesenteric arteries treated with 1μM MitoTempol, a superoxide dismutase mimetic and mitochondrial anti-oxidant, show a decreased responsiveness to BAY 58-2667 (n=3-5/group). Notably, we have published evidence that cytochrome b5 reductase 3 (CYB5R3) acts as an sGC heme reductase (Fe 3+ -> Fe 2+ ) in cultured vascular smooth muscle cells. We recently performed vasoreactivity studies in transgenic CYB5R3 overexpression (OE CYB5R3) mice and show these mice have a significantly decreased responsiveness to BAY 58-2667 compared to controls irrespective of vascular bed (n=4-6/ group). Additionally, OE CYB5R3 mice exhibit a ~7mmHg decrease in pulse pressure and impaired blood pressure reduction in response to an acute injection of BAY 58-2667 as compared to controls (n=5/ group). Combined, these data indicate that mitochondria derived-reactive oxygen species and expression of CYB5R3 serve as a rheostat for modulating sensitivity to sGC activator BAY 58-2667 and vascular responsiveness in resistance arteries. Importantly, these results provide valuable insight into sGC activator therapy for treatment of hypertension and other cardiovascular related diseases.

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.

Endoplasmic reticulum stress inhibition blunts the development of essential hypertension in the spontaneously hypertensive rat.

Essential hypertension is the leading cause of premature death worldwide. However, hypertension's cause remains uncertain. endoplasmic reticulum (ER) stress has recently been associated with hypertension, but it is unclear whether ER stress causes hypertension. To clarify this question, we examined if ER stress occurs in blood vessels before the development of hypertension and if ER stress inhibition would prevent hypertension development. We used the spontaneously hypertensive rat (SHR) as a model of human essential hypertension and the Wistar-Kyoto (WKY) rat as its normotensive control. Resistance arteries collected from young rats determined that ER stress was present in SHR vessels before the onset of hypertension. To assess the effect of ER stress inhibition on hypertension development, another subset of rats were treated with 4-phenylbutyric acid (4-PBA; 1 g·kg-1·day-1) for 8 wk from 5 wk of age. Blood pressure was measured via radiotelemetry and compared with untreated SHR and WKY rats. Mesenteric resistance arteries were collected and assessed for structural and functional changes associated with hypertension. Systolic and diastolic blood pressures were significantly lower in the 4-PBA-treated SHR groups than in untreated SHRs. Additionally, 4-PBA significantly decreased the media-to-lumen ratio and ER stress marker expression, improved vasodilatory response, and reduced contractile responses in resistance arteries from SHRs. Overall, ER stress inhibition blunted the development of hypertension in the SHR. These data add evidence to the hypothesis that a component of hypertension in the SHR is caused by ER stress. NEW & NOTEWORTHY In this study, 4-phenylbutyric acid's (4-PBA's) molecular chaperone capability was used to inhibit endoplasmic reticulum (ER) stress in the small arteries of young spontaneously hypertensive rats (SHRs) and reduce their hypertension. These effects are likely mediated through 4-PBA's effects to reduce resistant artery contractility and increase nitric oxide-mediated endothelial vasodilation through a process preventing endothelial dysfunction. Overall, ER stress inhibition blunted the development of hypertension in this young SHR model. This suggests that a component of the increase in blood pressure found in SHRs is due to ER stress. However, it is important to note that inhibition of ER stress was not able to fully restore the blood pressure to normal, suggesting that a component of hypertension may not be due to ER stress. This study points to the inhibition of ER stress as an important new physiological pathway to lower blood pressure, where other known approaches may not achieve blood pressure-lowering targets.

Increased Contractile Response to Noradrenaline Induced By Factors Associated with the Metabolic Syndrome in Cultured Small Mesenteric Arteries

This study investigated the effect of the metabolic syndrome associated risk factors hyperglycemia (glucose [Glc]), hyperinsulinemia (insulin [Ins]) and low-grade inflammation (tumor necrosis factor α [TNFα]) on the vasomotor responses of resistance arteries. Isolated small mesenteric arteries from 3-month-old Sprague-Dawley rats, were suspended for 21-23 h in tissue cultures containing either elevated Glc (30 mmol/l), Ins (100 nmol/l), TNFα (100 ng/ml) or combinations thereof. After incubation, the vascular response to noradrenaline (NA), phenylephrine, isoprenaline and NA in the presence of propranolol (10 µmol/l) was measured by wire myography. Results: Arteries exposed only to combinations of the risk factors showed a significant 1.6-fold increase in the contractile NA sensitivity, which suggests that complex combinations of metabolic risk factors might lead to changes in vascular tone.

Abstract 18266: Overactive Tgfβ Signaling in Smooth Muscle Cells Induces Hypertension in Mice Through Increased Myogenic Tone and is Responsible for Increased Myogenic Tone in Resistance Vessels of Hypertensive Patients

Emilin1 (E1) is a protein of the extracellular matrix that regulates TGFβ activity through proteolysis of the proTGFβ. E1 KO mice are hypertensive, with increased TGFβ activation. As E1 is expressed in blood vessels starting from embryonic life to adulthood, is still unknown whether the E1 KO phenotype results from a developmental defect or lack of a homeostatic role exerted in the adult. To dissect this issue, we inactivated E1 in smooth muscle cells (VSMCs) of adult mice, by the use of floxed E1 and CreERT2 [a tamoxifen (TAM) inducible Cre recombinase] under the control of the smooth muscle myosin heavy chain (Smmhc) promoter. When Smmhc-CreERT2 E1fl/fl mice were given TAM, blood pressure significantly increased (124±1 vs basal condition 106±1 mmHg) as well as myogenic response in resistance arteries (16.3±0.7 vs basal condition 11.4±0.1 % at 125 mmHg). In order to evaluate the relevance of our findings in the human pathology, we enrolled 20 hypertensive and 20 normotensive patients that underwent neurosurgical intervention for primary lumbar hernia repair and dissected out resistance vessels from skeletal muscle biopsies. We analyzed the myogenic tone of human peripheral resistance vessels in a pressure myograph and randomized vessels to receive vehicle, a neutralizing antibody anti-TGFβ or a preimmune non relevant IgG. As first, we found that hypertensive patients had significantly increased myogenic tone as compared to normotensive (16.6±2.1% vs 10.5±0.4% at 125 mmHg). Moreover, the pretreatment of vessels with anti-TGFβ antibody normalized the myogenic tone of hypertensive patients (10.8±0.6%) to the levels of normotensives (11.8±1.3%). A pretreatment with the non relevant IgG did not modify the myogenic tone of hypertensives (17±0.6%), thus demonstrating that the effect was dependent of an increased TGFβ signaling in vessels of hypertensive patients. Overall, our results suggest that the TGFβ pathway in VSMC, is crucial for the myogenic tone of resistance arteries and is linked to the regulation of blood pressure. The data obtained in peripheral resistance vessels from hypertensive patients, that display an increased myogenic tone dependent on overactive TGFβ signaling, highlight the translational potential of our findings to the human pathology.

Abstract 051: The Myogenic Tone Of Resistance Arteries Is Controlled By A Molecular Pathway Driven By Tgfβ Through Emilin1 And Converging On The Regulation Of Trp Channels.

Emilin1 (E1) is a protein of the extracellular matrix that regulates TGFβ bioavailability through proteolysis of the proTGFβ. E1 KO mice are hypertensive, with increased TGFβ activation. As E1 is expressed in blood vessels starting from embryonic life to adulthood, is still unknown whether the E1 KO phenotype results from a developmental defect or lack of a homeostatic role in the adult. To dissect this issue, we used a conditional gene targeting approach to inactivate E1 in smooth muscle cells (VSMCs) of adult mice, by the use of floxed E1 and CreERT2 [tamoxifen (TAM) inducible Cre recombinase] under the control of the smooth muscle myosin heavy chain (Smmhc) promoter. When E1fl/fl mice carrying the Smmhc-CreERT2 were given TAM blood pressure significantly increased (124±1 vs basal condition 106±1 mmHg) as well as myogenic response in resistance arteries (16.3±0.7 vs basal condition 11.4±0.1 % at 125 mmHg). In order to understand how E1 ablation in VSMCs could functionally determine the increase in myogenic tone, we used an ex vivo approach in resistance arteries, to target the molecular pathway that could regulate intracellular calcium fluxes, the main regulator of myogenic contraction. Experiments performed in isolated VSMCs from E1 KO as compared to WT arteries, suggested the involvement of a ROCE (receptor operated calcium entry) mechanism, whose main final molecular determinant is represented by the TRP channels, known to be regulated by a canonical PLC-DAG pathway, but also by a parallel one controlled by the growth factors signaling cascade. By exploring this pathway, we found that E1 regulates the activity of TRPC3 channels and thus of the myogenic response. The molecular pathway delineated by our studies started from the transactivation of EGFR through metalloproteinases and converged on the Src kinases that increased TRPC3 function and thus calcium entry. In particular, with an in vivo targeting of TRPC3 in E1 KO with an adenovirus carrying a dominant negative mutation, we demonstrated a rescue of the increased myogenic response of E1 KO resistance arteries. Overall our results suggest that this pathway in VSMC, driven by TGFβ and regulated by E1, is crucial for the myogenic tone of resistance arteries and is linked to the regulation of blood pressure.

Effects of glucagon-like peptide-1 in diabetic rat small resistance arteries.

We investigated the functional effects of glucagon-like peptide-1 [GLP-1(7-36)] and GLP-1(9-36) and the mechanism(s) playing a role in the effects of these agents in isolated small resistance arteries from control and diabetic rats. Cumulative concentrations of GLP-1(7-36) and GLP-1(9-36) produced concentration-dependent relaxations in endothelium-intact but not endothelium-denuded arteries that were significantly decreased in diabetic rats. GLP-1 receptor antagonist exendin(9-39) significantly inhibited responses to GLP-1 analogs. Nitric oxide/cyclic guanosine monophosphate pathway blockers, but not indomethacin, significantly decreased responses to GLP-1(7-36) or GLP-1(9-36) in control and diabetic rats. 4-Aminopyridine or glibenclamide incubation did not alter relaxations to GLP-1 analogs. GLP-1(7-36)- and GLP-1(9-36)-induced relaxations were blunted significantly and to similar extends by charybdotoxin and apamin combination in control and diabetic rats. Catalase did not affect, whereas superoxide dismutase (SOD) caused a significant increase in relaxations to GLP-1 analogs only in diabetic rats. We provided evidence about the relaxant effects of GLP-1(7-36) and GLP-1(9-36) in resistance arteries that were reduced in diabetic rats. Both calcium-activated potassium channels and endothelium played a major role in relaxations. Increment in certain reactive oxygen species and/or reduction in superoxide dismutase function might play a role in reduced relaxant responses of resistance arteries to GLP-1(7-36) and GLP-1(9-36) in diabetic rats.

Abstract 511: Emilin1 Controls Myogenic Response of Resistance Arteries by Modulation of TRPC6 Through the TGFß/ALK5/ADAM17/EGFR Pathway

Emilin1 (E1), a protein of the extracellular matrix expressed in the cardiovascular system, regulates TGFβ bioavailability through proteolysis of the proTGFβ, that releases the growth factor. E1 null mice are hypertensive, with increased TGFβ activation. As E1 is expressed in blood vessels starting from embryonic life to adulthood, is still unknown whether the E1 null phenotype results from a developmental defect or lack of a homeostatic role exerted during the adult life. To dissect this issue, we used a conditional gene targeting approach to inactivate E1 in smooth muscle cells (SMCs) of adult mice, by the use of floxed E1 and CreER T2 [a tamoxifen (TAM) inducible Cre recombinase] under the control of the smooth muscle myosin heavy chain (Smmhc) promoter. TAM induced Cre activity selectively in SMCs, as shown in mice with the Rosa26R mutation. When E1 fl/fl mice carrying the Smmhc-CreER T2 (Smmhc-CreER T2 E1 fl/fl ) were given TAM, E1 protein completely disappeared and blood pressure significantly increased, as compared to E1 fl/fl . While no structural changes of arteries were detected, we found that mesenteric arteries of Smmhc-CreER T2 E1 fl/fl displayed an increased myogenic response to pressure as compared to E1 fl/fl . Since the hypertensive phenotype of Smmhc-CreER T2 E1 fl/fl mice was rescued by a treatment with a TGFβ neutralizing antibody, we investigated the molecular mechanism in SMCs that, downstream to this pathway, could lead to an increased Ca 2+ -mediated contractility. In particular, also the increased myogenic response was rescued by exposure of resistance arteries to a TGFβ neutralizing antibody and, more important, by the inhibition of TGFβ receptor ALK5, obtained by in vivo vessels transfection with an adenovirus carrying a dominant negative ALK5 mutant. Moreover, we found that the increased TGFβ signaling, by phosphorylating the ADAM17/EGFR cascade, converged on overactivation of the Transient receptor potential cation channel C6 (TRPC6), rescued by in vivo vessels transfection with an adenovirus carrying a dominant negative TRPC6 mutant. Our results demonstrate that E1 exerts a homeostatic control of myogenic response by regulating the TRPC6 Ca 2+ channel, through a modulation of the TGFβ/ALK5/ADAM17/EGFR signaling pathway in SMCs.

Toll‐like receptor 2 activation increases adrenergic sensitivity in mesenteric resistance vessels of rats

Toll-like receptors (TLRs) are pattern recognition receptors of the innate immune system that recognize microbial pathogens as well as endogenous damage-associated molecular patterns (DAMPS). TLR2 plays a role in cardiovascular diseases (CVD) such as atherosclerosis and heart failure, but its role in vascular function is unknown. We hypothesized that TLR2 activation would increase contractile responses in resistance arteries. We used second order mesenteric arteries from 15-week old Sprague Dawley rats in a small vessel wire myograph. Concentration response curves to norepinephrine (NE; 10−9–10−4 M) were performed in vessels incubated with and without TLR2 agonist Pam3CSK4 [(P3C4); 300ng/mL; 35′ prior incubation], as well as a combination of P3C4 and TLR2 antibody (1 μg/mL; 35′ prior incubation). The sensitivity of the vessels to NE was increased in vessels treated with P3C4 (LogEC50: 6.13± 0.08) compared to control (LogEC50: 5.55± 0.09), and this increase was attenuated by anti-TLR2 (LogEC50: 5.53± 0.05) (p<.05). These results indicate that TLR2 activation increases the sensitivity to adrenergic stimuli in resistance vessels, potentially leading to vascular dysfunction and contributing to CVD such as hypertension.

Differential effects of aging and exercise training on intra‐abdominal adipose arteriolar function and blood flow regulation

During exercise, an old age‐associated inability to increase vascular resistance within intra‐abdominal adipose tissue (AT) may compromise the ability to redistribute blood flow. We tested the hypotheses that 1) there would be an elevated perfusion of AT during exercise with old age, associated with diminished arterial vasoconstrictor responses, and 2) exercise training (ET) would mitigate age‐associated alterations in AT blood flow. Young (Y, 6 mo; n=40) and old (O, 24 mo; n=28) male Fischer 344 rats were divided into sedentary (YSed, OSed) or ET (YET, OET, 10wks treadmill exercise) groups. Blood flow at rest and during exercise and α‐adrenergic and myogenic vasoconstrictor responses in resistance arteries from AT were measured. During exercise there was a directionally opposite AT blood flow response in OSed group (~150% increase) and YSed (~ 55% decrease) versus resting values. Both α‐adrenergic and myogenic vasoconstriction were diminished in OSed versus YSed. ET resulted in a similar AT hyperemic response between age groups during exercise (YET, 10 ± 1; OET, 8 ± 1 ml/min/100 g) and was associated with enhanced myogenic and α‐adrenergic vasoconstriction in AT arteries from the OET group relative to OSed. These results indicate that there is an inability to increase vascular resistance in AT during exercise with old age due, in part, to a diminished vasoconstriction in AT arteries.

Deficiency in caveolin‐1 or type 2 diabetes augments vasoconstrictor responses to sphingosine‐1‐phosphate

Sphingolipids are not only integral membrane components but also vasoactive signaling molecules. We tested the hypothesis that vascular responses to sphingosine‐1‐phosphate (S1P) are augmented in diabetics through abnormal rho kinase and caveolar regulation. We measured responses of isolated pressurized resistance arteries from gracilis muscle of wild type (WT, diameter 101±23μm), caveolin‐1 deficient which lack caveolae (Cav−/−, diameter 130±30μm) and a model of type 2 diabetes in mice [leptin receptor deficient (db/db), diameter 123±11μm] to S1P (1nM‐1μM) and KCl (25–100 mM) in the absence and presence of a rho kinase inhibitor, H1152 (1 μM). S1P produced concentration‐dependent vasoconstriction that was increased 60–70% (P&lt;0.05) in arteries from Cav−/− and db/db mice. H1152 had minimal effect on constriction of arteries from WT mice but inhibited responses in arteries from Cav−/− and db/db mice to the levels of WT. Vasoconstriction to KCl was similar in all groups. We conclude that in type 2 diabetics, responses of resistance arteries in skeletal muscle to S1P are augmented. The increased constriction may be due, in part, to abnormal caveolar localization and/or rho kinase activation. Support: Dept of Veterans Affairs

Ca2+ sensitization due to myosin light chain phosphatase inhibition and cytoskeletal reorganization in the myogenic response of skeletal muscle resistance arteries

Abstract The myogenic response of resistance arteries to intravascular pressure elevation is a fundamental physiological mechanism of crucial importance for blood pressure regulation and organ-specific control of blood flow. The importance of Ca(2+) entry via voltage-gated Ca(2+) channels leading to phosphorylation of the 20 kDa myosin regulatory light chains (LC20) in the myogenic response is well established. Recent studies, however, have suggested a role for Ca(2+) sensitization via activation of the RhoA/Rho-associated kinase (ROK) pathway in the myogenic response. The possibility that enhanced actin polymerization is also involved in myogenic vasoconstriction has been suggested. Here, we have used pressurized resistance arteries from rat gracilis and cremaster skeletal muscles to assess the contribution to myogenic constriction of Ca(2+) sensitization due to: (1) phosphorylation of the myosin targeting subunit of myosin light chain phosphatase (MYPT1) by ROK; (2) phosphorylation of the 17 kDa protein kinase C (PKC)-potentiated protein phosphatase 1 inhibitor protein (CPI-17) by PKC; and (3) dynamic reorganization of the actin cytoskeleton evoked by ROK and PKC. Arterial diameter, MYPT1, CPI-17 and LC20 phosphorylation, and G-actin content were determined at varied intraluminal pressures ± H1152, GF109203X or latrunculin B to suppress ROK, PKC and actin polymerization, respectively. The myogenic response was associated with an increase in MYPT1 and LC20 phosphorylation that was blocked by H1152. No change in phospho-CPI-17 content was detected although the PKC inhibitor, GF109203X, suppressed myogenic constriction. Basal LC20 phosphorylation at 10 mmHg was high at ∼40%, increased to a maximal level of ∼55% at 80 mmHg, and exhibited no additional change on further pressurization to 120 and 140 mmHg. Myogenic constriction at 80 mmHg was associated with a decline in G-actin content by ∼65% that was blocked by inhibition of ROK or PKC. Taken together, our findings indicate that two mechanisms of Ca(2+) sensitization (ROK-mediated phosphorylation of MYPT1-T855 with augmentation of LC20 phosphorylation, and a ROK- and PKC-evoked increase in actin polymerization) contribute to force generation in the myogenic response of skeletal muscle arterioles.

Differential effects of aging and exercise on intra-abdominal adipose arteriolar function and blood flow regulation

Adipose tissue (AT), which typically comprises an increased percentage of body mass with advancing age, receives a large proportion of resting cardiac output. During exercise, an old age-associated inability to increase vascular resistance within the intra-abdominal AT may compromise the ability of the cardiovascular system to redistribute blood flow to the active musculature, contributing to the decline in exercise capacity observed in this population. We tested the hypotheses that 1) there would be an elevated perfusion of AT during exercise with old age that was associated with diminished vasoconstrictor responses of adipose-resistance arteries, and 2) chronic exercise training would mitigate the age-associated alterations in AT blood flow and vascular function. Young (6 mo; n = 40) and old (24 mo; n = 28) male Fischer 344 rats were divided into young sedentary (YSed), old sedentary (OSed), young exercise trained (YET), or old exercise trained (OET) groups, where training consisted of 10-12 wk of treadmill exercise. In vivo blood flow at rest and during exercise and in vitro α-adrenergic and myogenic vasoconstrictor responses in resistance arteries from AT were measured in all groups. In response to exercise, there was a directionally opposite change in AT blood flow in the OSed group (≈ 150% increase) and YSed (≈ 55% decrease) vs. resting values. Both α-adrenergic and myogenic vasoconstriction were diminished in OSed vs. YSed AT-resistance arteries. Exercise training resulted in a similar AT hyperemic response between age groups during exercise (YET, 9.9 ± 0.5 ml · min(-1) · 100(-1) g; OET, 8.1 ± 0.9 ml · min(-1) · 100(-1) g) and was associated with enhanced myogenic and α-adrenergic vasoconstriction of AT-resistance arteries from the OET group relative to OSed. These results indicate that there is an inability to increase vascular resistance in AT during exercise with old age, due, in part, to a diminished vasoconstriction of AT arteries. Furthermore, the results indicate that exercise training can augment vasoconstriction of AT arteries and mitigate age-related alterations in the regulation of AT blood flow during exercise.

Vascular Smooth Muscle Emilin-1 Is a Regulator of Arteriolar Myogenic Response and Blood Pressure

Emilin-1 is a protein of elastic extracellular matrix involved in blood pressure (BP) control by negatively affecting transforming growth factor (TGF)-β processing. Emilin1 null mice are hypertensive. This study investigates how Emilin-1 deals with vascular mechanisms regulating BP. This study uses a phenotype rescue approach in which Emilin-1 is expressed in either endothelial cells or vascular smooth muscle cells of transgenic animals with the Emilin1(-/-) background. We found that normalization of BP required Emilin-1 expression in smooth muscle cells, whereas expression of the protein in endothelial cells did not modify the hypertensive phenotype of Emilin1(-/-) mice. We also explored the effect of treatment with anti-TGF-β antibodies on the hypertensive phenotype of Emilin1(-/-) mice, finding that neutralization of TGF-β in Emilin1 null mice normalized BP quite rapidly (2 weeks). Finally, we evaluated the vasoconstriction response of resistance arteries to perfusion pressure and neurohumoral agents in different transgenic mouse lines. Interestingly, we found that the hypertensive phenotype was coupled with an increased arteriolar myogenic response to perfusion pressure, while the vasoconstriction induced by neurohumoral agents remained unaffected. We further elucidate that, as for the hypertensive phenotype, the increased myogenic response was attributable to increased TGF-β activity. Our findings clarify that Emilin-1 produced by vascular smooth muscle cells acts as a main regulator of resting BP levels by controlling the myogenic response in resistance arteries through TGF-β.

Pioglitazone treatment increases COX‐2‐derived prostacyclin production and reduces oxidative stress in hypertensive rats: role in vascular function

PPARγ agonists, glitazones, have cardioprotective and anti-inflammatory actions associated with gene transcription interference. In this study, we determined whether chronic treatment of adult spontaneously hypertensive rats (SHR) with pioglitazone alters BP and vascular structure and function, and the possible mechanisms involved. Mesenteric resistance arteries from untreated or pioglitazone-treated (2.5 mg·kg⁻¹ ·day⁻¹ , 28 days) SHR and normotensive [Wistar Kyoto (WKY)] rats were used. Vascular structure was studied by pressure myography, vascular function by wire myography, protein expression by Western blot and immunohistochemistry, mRNA levels by RT-PCR, prostanoid levels by commercial kits and reactive oxygen species (ROS) production by dihydroethidium-emitted fluorescence. In SHR, pioglitazone did not modify either BP or vascular structural and mechanical alterations or phenylephrine-induced contraction, but it increased vascular COX-2 levels, prostacyclin (PGI₂) production and the inhibitory effects of NS 398, SQ 29,548 and tranylcypromine on phenylephrine responses. The contractile phase of the iloprost response, which was reduced by SQ 29,548, was greater in pioglitazone-treated and pioglitazone-untreated SHR than WKY. In addition, pioglitazone abolished the increased vascular ROS production, NOX-1 levels and the inhibitory effect of apocynin and allopurinol on phenylephrine contraction, whereas it did not modify eNOS expression but restored the potentiating effect of N-nitro-L-arginine methyl ester on phenylephrine responses. Although pioglitazone did not reduce BP in SHR, it increased COX-2-derived PGI₂ production, reduced oxidative stress, and increased NO bioavailability, which are all involved in vasoconstrictor responses in resistance arteries. These effects would contribute to the cardioprotective effect of glitazones reported in several pathologies.

Raloxifene improves vascular reactivity in pressurized septal coronary arteries of ovariectomized hamsters fed cholesterol diet

Although vascular effects of selective estrogen receptor modulators (SERMs) have been extensively examined in conduit arteries, whether SERMs could favorably modulate myogenic response in resistance arteries is unknown. The impact of raloxifene therapy and cholesterol diet on myogenic constriction during estrogen deficiency is unresolved. This study investigated changes in vascular reactivity and myogenic responses in female ovariectomized (Ovx) hamsters fed high-cholesterol diet (HCD) with and without chronic treatment of raloxifene. Functional studies were performed on hamster septal coronary arteries cannulated in a pressure myograph. Acetylcholine (ACh)-induced dilatation was reduced in arteries from cholesterol-fed Ovx hamsters, but not in those from cholesterol-fed hamsters, while pressure-induced myogenic constriction was unaffected. Chronic treatment with raloxifene restored ACh-induced dilatation in cholesterol-fed Ovx hamsters. U46619-induced constriction was increased in arteries from cholesterol-fed Ovx hamsters but not from cholesterol-fed control hamsters, which was normalized by chronic raloxifene treatment. The pressure–diameter relationship is presented as normalized diameter versus intraluminal pressure, while the effect of ACh or U46619 is expressed as percentage of tone at 80mmHg. Two-way analysis of variance (ANOVA) followed by Bonferroni post-tests were used for statistical evaluation among different treatment groups. P<0.05 was taken as statistically significant. The present results show that chronic treatment with raloxifene could benefit myogenically active coronary arteries by (i) restoring ACh-induced dilatation and (ii) reducing U46619-induced constriction without affecting pressure-induced myogenic responses in cholesterol-fed hamsters during estrogen deficiency. If such benefit can be observed in humans, raloxifene and other SERMs may be useful to preserve endothelial function and curtail vascular hypersensitivity in resistance coronary arteries in post-menopausal women with hypercholesterolemia or hyperlipidemia, a lipid condition implicated in the pathogenesis of myocardial infarction.

Myogenic responsiveness in resistance arteries of diabetic mice is enhanced via a TNFα/S1P‐mediated mechanism

Diabetes is associated with structural and functional microcirculatory changes that impair the ability of resistance arteries to autoregulate tissue perfusion and blood pressure. Autoregulation relies on the myogenic response (MR), an intrinsic property of vascular smooth muscle cells (VSMC). We have identified vascular sphingosine-1-phosphate (S1P2) signalling as an essential component of the MR that can be activated by tumor necrosis factor α (TNFα). As both high-glucose treatment and diabetes are associated with upregulation of VSMC TNFα, we examined TNFα as a putative mediator of diabetes-associated changes in myogenic responsiveness. MRs were assessed in mouse mesenteric arteries after induction of diabetes by high fat diet (HFD)/streptozotocin protocol. MRs were significantly augmented in diabetic mice compared to control (HFD-only) mice, while responses to phenylephrine were similar in both groups. Both the TNFα scavenger etanercept and the S1P2 receptor antagonist JTE013 abolished increased MRs in diabetic mice with fully maintained responses to phenylephrine. Therefore, our data suggests that TNFα regulates MR changes in diabetes through the involvement of S1P signalling. Funding: NSERC/HSFC

Ca 2+ Sensitization Contributes to Myogenic Control of Arterial Diameter in Skeletal Muscle Resistance Arteries

The myogenic response of resistance arteries to changes in intravascular pressure is a crucial determinant of peripheral vascular resistance. Ca2+ sensitization via inhibition of myosin light chain phosphatase (MLCP), due to phosphorylation of the MLCP targeting subunit 1 (MYPT1) by Rho-associated kinase (ROK), contributes to myogenic constriction of cerebral arteries. As it is unknown if this occurs in non-cerebral vessels we assessed the role of Ca2+ sensitization in the myogenic response of skeletal muscle arterioles (rat gracilis and cremaster). ROK inhibition with H1152 suppressed myogenic constriction between 40 and 120 mmHg. Increasing pressure from 10 to 80 mmHg caused an H1152-sensitive increase in MYPT1-T855 and myosin regulatory light chain (LC20) phosphorylation, but phospho-MYPT1-T697 and -CPI-17 levels were unchanged. Gracilis and cremaster arteries showed high basal levels of LC20 phosphorylation at 10 mmHg, comparable to the level in cerebral arteries at 60 mmHg. These findings suggest that Ca2+ sensitization contributes to the myogenic response of skeletal muscle resistance arteries. High basal LC20 phosphorylation may be the result of increased basal MYPT1 phosphorylation and indicates that myogenic constriction occurs over a narrow range of LC20 phosphorylation. (Support from CIHR MOP-97988 to WCC, AHFMR/CIHR to AEY and Spanish Ministry of Education & Science to OC).

Potential involvement of actin cytoskeleton reorganization in generation of the arterial myogenic response

The myogenic response of resistance arteries to intravascular pressure is due to cellular processes intrinsic to vascular smooth muscle cells. Myogenic constriction of cerebral arteries involves the activation and inhibition of myosin light chain kinase and phosphatase, respectively, to increase myosin light chain phosphorylation (pLC20) and permit cross‐bridge cycling. However, equivalent levels of pLC20 are detected at 80 mmHg when pressurized vessels flash‐frozen prior to or after spontaneous development of myogenic tone during the equilibration period are compared. Also, myogenic constriction evoked by a pressure step to 80 mmHg is observed following a maximal increase in pLC20 (~50%) due to 5‐HT stimulation, but there is no further increase in pLC20, or change in p‐caldesmon or p‐calponin. Latrunculin B (10 μM), that binds free G‐actin, had no effect on constriction due to 5‐HT or 80 mmHg alone, but it reversed contractions due to the combination of 5‐HT and 80 mmHg, or pressure alone at 120 mmHg, that are associated with maximal levels of pLC20. Our findings indicate that re‐establishment of myofilament‐actin cytoskeleton contacts during equilibration is necessary for myogenic constriction, and dynamic reorganization of the cytoskeleton may be required to faciltiate increased force generation in the myogenic response when peak levels of pLC20 are attained. (Supported by CIHR MOP‐97988 and AIHS).