Pressure Myography Research Articles

Carotid artery stenosis in hypertensive rats impairs dilatory pathways in parenchymal arterioles.

Hypertension is a leading risk factor for vascular cognitive impairment and is strongly associated with carotid artery stenosis. In normotensive rats, chronic cerebral hypoperfusion induced by bilateral common carotid artery stenosis (BCAS) leads to cognitive impairment that is associated with impaired endothelium-dependent dilation in parenchymal arterioles (PAs). The aim of this study was to assess the effects of BCAS on PA function and structure in stroke-prone spontaneously hypertensive rats, a model of human essential hypertension. Understanding the effects of hypoperfusion on PAs in a hypertensive model could lead to the identification of therapeutic targets for cognitive decline in a model that reflects the at-risk population. We hypothesized that BCAS would impair endothelium-dependent dilation in PAs and induce artery remodeling compared with sham rats. PAs from BCAS rats had endothelial dysfunction, as assessed using pressure myography. Inhibition of nitric oxide and prostaglandin production had no effect on PA dilation in sham or BCAS rats. Surprisingly, inhibition of epoxyeicosatrienoic acid production increased dilation in PAs from BCAS rats but not from sham rats. Similar results were observed in the presence of inhibitors for all three dilatory pathways, suggesting that epoxygenase inhibition may have restored a nitric oxide/prostaglandin-independent dilatory pathway in PAs from BCAS rats. PAs from BCAS rats underwent remodeling with a reduced wall thickness. These data suggest that marked endothelial dysfunction in PAs from stroke-prone spontaneously hypertensive rats with BCAS may be associated with the development of vascular cognitive impairment. NEW & NOTEWORTHY The present study assessed the structure and function of parenchymal arterioles in a model of chronic cerebral hypoperfusion and hypertension, both of which are risk factors for cognitive impairment. We observed that impaired dilation and artery remodeling in parenchymal arterioles and abolished cerebrovascular reserve capacity may mediate cognitive deficits.

Cerebrovascular recovery after stroke with individual and combined losartan and captopril treatment of SHRsp

We assessed whether the superior restoration of cerebrovascular function after hemorrhagic stroke by losartan versus captopril treatment was due to better BP, uremia, uricaemia, or aldosterone control in Kyoto Wistar stroke-prone-hypertensive rats and evaluated whether elevated angiotensin II (A2) levels enhanced the effectiveness of losartan treatment. Constriction was studied in the middle cerebral arteries (MCAs) using a pressure myograph. Post-stroke survival increased from 21 to 310 and 189days respectively with losartan and captopril treatment. Neither treatment reduced BP, both reversed uremia and hyperaldosteronism equally after 7days. Plasma uric acid remained low. At stroke, MCA constriction to pressure (PDC), protein kinase C (PKC) activation, depolarization, and sarcoplasmic Ca2+ were attenuated. Endothelial-dependent-vasodilation by bradykinin and endogenous NO release were lost. Both treatments recovered these functions within 7days. These functions deteriorated after 116days of captopril but not losartan treatment. Inhibiting A2 formation during losartan treatment didn't alter BP or vascular recovery. The superior recovery of PDC by losartan over captopril was not produced by better BP, uremia or aldosterone control or elevated A2. PDC recovery was associated with improved PKC function and enhanced basal NO release. The re-establishment of PDC could reduce cerebrovascular over-perfusion and hematoma expansion after stroke.

Hyperoxia does not directly affect vascular tone in isolated arteries from mice.

Hospitalized patients often receive oxygen supplementation, which can lead to a supraphysiological oxygen tension (hyperoxia). Hyperoxia can have hemodynamic effects, including an increase in systemic vascular resistance. This increase suggests hyperoxia-induced vasoconstriction, yet reported direct effects of hyperoxia on vessel tone have been inconsistent. Furthermore, hyperoxia-induced changes in vessel diameter have not been studied in mice, currently the most used mammal model of disease. In this study we set out to develop a pressure-myograph model using isolated vessels from mice for investigation of pathways involved in hyperoxic vasoconstriction. Isolated conduit and resistance arteries (femoral artery and gracilis arteriole, respectively) from C57BL/6 mice were exposed to normoxia (PO2 of 80 mmHg) and three levels of hyperoxia (PO2 of 215, 375 and 665 mmHg) in a no-flow pressure myograph setup. Under the different PO2 levels, dose-response agonist induced endothelium-dependent vasodilation (acetylcholine, arachidonic acid), endothelium-independent vasodilation (s-nitroprusside), as well as vasoconstriction (norepinephrine, prostaglandin F2α) were examined. The investigated arteries did not respond to oxygen by a change in vascular tone. In the dose-response studies, maximal responses and EC50 values to any of the aforementioned agonists were not affected by hyperoxia either. We conclude that arteries and arterioles from healthy mice are not intrinsically sensitive to hyperoxic conditions. The present ex-vivo model is therefore not suitable for further research into mechanisms of hyperoxic vasoconstriction.

Reduced expression of PMCA1 is associated with increased blood pressure with age which is preceded by remodelling of resistance arteries

SummaryHypertension is a well‐established risk factor for adverse cardiovascular events, and older age is a risk factor for the development of hypertension. Genomewide association studies have linked ATP2B1, the gene for the plasma membrane calcium ATPase 1 (PMCA1), to blood pressure (BP) and hypertension. Here, we present the effects of reduction in the expression of PMCA1 on BP and small artery structure and function when combined with advancing age. Heterozygous PMCA1 null mice (PMCA1Ht) were generated and conscious BP was measured at 6 to 18 months of age. Passive and active properties of isolated small mesenteric arteries were examined by pressure myography. PMCA1Ht mice exhibited normal BP at 6 and 9 months of age but developed significantly elevated BP when compared to age‐matched wild‐type controls at ≥12 months of age. Decreased lumen diameter, increased wall thickness and increased wall:lumen ratio were observed in small mesenteric arteries from animals 9 months of age and older, indicative of eutrophic remodelling. Increases in mesenteric artery intrinsic tone and global intracellular calcium were evident in animals at both 6 and 18 months of age. Thus, decreased expression of PMCA1 is associated with increased BP when combined with advancing age. Changes in arterial structure precede the elevation of BP. Pathways involving PMCA1 may be a novel target for BP regulation in the elderly.

Short-term (48hours) intravenous serelaxin infusion has no effect on myogenic tone or vascular remodeling in rat mesenteric arteries.

Short-term IV sRLX (recombinant human relaxin-2) infusion enhances endothelium-dependent relaxation in mesenteric arteries. This is initially underpinned by increased NO followed by a transition to prostacyclin. The effects of short-term IV sRLX treatment on pressure-induced myogenic tone and vascular remodeling in these arteries are unknown. Therefore, we investigated the effects of sRLX infusion on pressure-induced myogenic tone and passive mechanical wall properties in mesenteric arteries. Mesenteric artery myogenic tone and passive mechanics were examined after 48-hours and 10-days infusion of sRLX. Potential mechanisms of action were assessed by pressure myography, qPCR, and Western blot analysis. Neither 48-hours nor 10-days sRLX treatment had significant effects on myogenic tone, passive arterial wall stiffness, volume compliance, or axial lengthening. However, in 48-hours sRLX -treated rats, incubation with the NO synthase blocker L-NAME significantly increased myogenic tone (P<.05 vs placebo), demonstrating an increased contribution of NO to the regulation of myogenic tone. eNOS dimerization, but not phosphorylation, was significantly upregulated in the arteries of sRLX -treated rats. In mesenteric arteries, 48-hours sRLX treatment upregulates the role of NO in the regulation of myogenic tone by enhancing eNOS dimerization, without altering overall myogenic tone or vascular remodeling.

Imaging and modeling of acute pressure-induced changes of collagen and elastin microarchitectures in pig and human resistance arteries.

The impact of disease-related changes in the extracellular matrix (ECM) on the mechanical properties of human resistance arteries largely remains to be established. Resistance arteries from both pig and human parietal pericardium (PRA) display a different ECM microarchitecture compared with frequently used rodent mesenteric arteries. We hypothesized that the biaxial mechanics of PRA mirror pressure-induced changes in the ECM microarchitecture. This was tested using isolated pig PRA as a model system, integrating vital imaging, pressure myography, and mathematical modeling. Collagenase and elastase digestions were applied to evaluate the load-bearing roles of collagen and elastin, respectively. The incremental elastic modulus linearly related to the straightness of adventitial collagen fibers circumferentially and longitudinally (both R2 ≥ 0.99), whereas there was a nonlinear relationship to the internal elastic lamina elastin fiber branching angles. Mathematical modeling suggested a collagen recruitment strain (means ± SE) of 1.1 ± 0.2 circumferentially and 0.20 ± 0.01 longitudinally, corresponding to a pressure of ~40 mmHg, a finding supported by the vital imaging. The integrated method was tested on human PRA to confirm its validity. These showed limited circumferential distensibility and elongation and a collagen recruitment strain of 0.8 ± 0.1 circumferentially and 0.06 ± 0.02 longitudinally, reached at a distending pressure below 20 mmHg. This was confirmed by vital imaging showing negligible microarchitectural changes of elastin and collagen upon pressurization. In conclusion, we show here, for the first time in resistance arteries, a quantitative relationship between pressure-induced changes in the extracellular matrix and the arterial wall mechanics. The strength of the integrated methods invites for future detailed studies of microvascular pathologies.NEW & NOTEWORTHY This is the first study to quantitatively relate pressure-induced microstructural changes in resistance arteries to the mechanics of their wall. Principal findings using a pig model system were confirmed in human arteries. The combined methods provide a strong tool for future hypothesis-driven studies of microvascular pathologies.

Mechanical behavior and matrisome gene expression in the aneurysm-prone thoracic aorta of newborn lysyl oxidase knockout mice.

Mutations in lysyl oxidase (LOX) are associated with thoracic aortic aneurysm and dissection (TAAD). Mice that do not express Lox (Lox-/- ) die soon after birth and have 60% and 40% reductions in elastin- and collagen-specific cross-links, respectively. LOX inactivation could also change the expression of secreted factors, the structural matrix, and matrix-associated proteins that constitute the aortic matrisome. We hypothesized that absence of Lox will change the mechanical behavior of the aortic wall because of reduced elastin and collagen cross-linking and alter the expression levels of matrisome and smooth muscle cell (SMC) genes in a vascular location-specific manner. Using fluorescence microscopy, pressure myography, and gene set enrichment analysis, we visualized the microarchitecture, quantified the mechanical behavior, and examined matrisome and SMC gene expression from ascending aortas (AAs) and descending aortas (DAs) from newborn Lox+/+ and Lox-/- mice. Even though Lox-/- AAs and DAs have fragmented elastic laminae and disorganized SMCs, the unloaded outer diameter and wall thickness were similar to Lox+/+ AAs and DAs. Lox-/- AAs and DAs have altered opening angles, circumferential stresses, and circumferential stretch ratios; however, only Lox-/- AAs have increased pressurized diameters and tangent moduli. Gene set enrichment analysis showed upregulation of the extracellular matrix (ECM) regulator gene set in Lox-/- AAs and DAs as well as differential expression of secreted factors, collagens, ECM-affiliated proteins, ECM glycoproteins, and SMC cell cycle gene sets that depend on the Lox genotype and vascular location. These results provide insights into the local chemomechanical changes induced by Lox inactivation that may be important for TAAD pathogenesis.NEW & NOTEWORTHY Absence of lysyl oxidase (Lox) causes thoracic aortic aneurysms. The aortic mechanical behavior of Lox-/- mice is consistent with reduced elastin and collagen cross-linking but demonstrates vascular location-specific differences. Lox-/- aortas show upregulation of matrix remodeling genes and location-specific differential expression of other matrix and smooth muscle cell gene sets.

Heterogeneous Vascular Bed Responses to Pulmonary Titanium Dioxide Nanoparticle Exposure.

A growing body of research links engineered nanomaterial (ENM) exposure to adverse cardiovascular endpoints. The purpose of this study was to evaluate the impact of ENM exposure on vascular reactivity in discrete segments so that we may determine the most sensitive levels of the vasculature where these negative cardiovascular effects are manifest. We hypothesized that acute nano-TiO2 exposure differentially affects reactivity with a more robust impairment in the microcirculation. Sprague-Dawley rats (8–10 weeks) were exposed to nano-TiO2 via intratracheal instillation (20, 100, or 200 µg suspended per 250 µL of vehicle) 24 h prior to vascular assessments. A serial assessment across distinct compartments of the vascular tree was then conducted. Wire myography was used to evaluate macrovascular active tension generation specifically in the thoracic aorta, the femoral artery, and third-order mesenteric arterioles. Pressure myography was used to determine vascular reactivity in fourth- and fifth-order mesenteric arterioles. Vessels were treated with phenylephrine, acetylcholine (ACh), and sodium nitroprusside. Nano-TiO2 exposure decreased endothelium-dependent relaxation in the thoracic aorta and femoral arteries assessed via ACh by 53.96 ± 11.6 and 25.08 ± 6.36%, respectively. Relaxation of third-order mesenteric arterioles was impaired by 100 and 20 µg nano-TiO2 exposures with mean reductions of 50.12 ± 8.7 and 68.28 ± 8.7%. Cholinergic reactivity of fourth- and fifth-order mesenteric arterioles was negatively affected by nano-TiO2 with diminished dilations of 82.86 ± 12.6% after exposure to 200 µg nano-TiO2, 42.6 ± 12.6% after 100 µg nano-TiO2, and 49.4 ± 12.6% after 20 µg nano-TiO2. Endothelium-independent relaxation was impaired in the thoracic aorta by 34.05 ± 25% induced by exposure to 200 µg nano-TiO2 and a reduction in response of 49.31 ± 25% caused by 100 µg nano-TiO2. Femoral artery response was reduced by 18 ± 5%, while third-order mesenteric arterioles were negatively affected by 20 µg nano-TiO2 with a mean decrease in response of 38.37 ± 10%. This is the first study to directly compare the differential effect of ENM exposure on discrete anatomical segments of the vascular tree. Pulmonary ENM exposure produced macrovascular and microvascular dysfunction resulting in impaired responses to endothelium-dependent, endothelium-independent, and adrenergic agonists with a more robust dysfunction at the microvascular level. These results provide additional evidence of an endothelium-dependent and endothelium-independent impairment in vascular reactivity.

T-type voltage-gated Ca2+ channels do not contribute to the negative feedback regulation of myogenic tone in murine superior epigastric arteries.

T‐type voltage‐gated Ca2+ channels (CaV3.2 VGCC) have been hypothesized to control spontaneous transient outward currents (STOCs) through large‐conductance Ca2+‐activated K+ channels (BKCa), and contribute to the negative‐feedback regulation of myogenic tone. We tested this hypothesis in superior epigastric arteries (SEAs) isolated from male C57BL/6 mice. SEAs were isolated and enzymatically dissociated to obtain single smooth muscle cells (SMCs) for whole‐cell recording of paxilline‐sensitive (PAX, 1 μmol/L) STOCs at −30 mV, or cannulated and studied by pressure myography (80 cm H2O, 37°C). The CaV3.2 blocker Ni2+ (30 μmol/L) had no effect on STOC amplitude (20.1 ± 1.7 pA vs. 20.6 ± 1.7 pA; n = 12, P = 0.6), but increased STOC frequency (0.79 ± 0.15 Hz vs. 1.21 ± 0.22 Hz; n = 12, P = 0.02). Although Ni2+ produced concentration‐dependent constriction of isolated, pressurized SEAs (logEC50 = −5.8 ± 0.09; E max = 72 ± 5% constriction), block of BKCa with PAX had no effect on vasoconstriction induced by 30 μmol/L Ni2+ (in the absence of PAX = 66 ± 4% constriction vs. in the presence of 1 μmol/L PAX = 65 ± 4% constriction; n = 7, P = 0.06). In contrast to Ni2+, the nonselective T‐type blocker, mibefradil, produced only vasodilation (logEC50 = −6.9 ± 0.2; E max = 74 ± 8% dilation), whereas the putative T‐type blocker, ML218, had no significant effect on myogenic tone between 10 nmol/L and 10 μmol/L (n = 6–7, P = 0.59). Our data do not support a role for CaV3.2 VGCC in the negative‐feedback regulation of myogenic tone in murine SEAs and suggest that Ni2+ may constrict SEAs by means other than block of CaV3.2 VGCC.

Mechanical behavior of the abdominal aortic aneurysm assessed by biaxial tests in the rat xenograft model

This paper addresses the mechanical biaxial behavior of degraded arteries obtained by the rat xenograft model. For that, a pressure myograph was used to perform extension-inflation tests on abdominal aortic aneurysms (AAAs). Furthermore, residual stresses in the aneurismal wall were assessed by opening angle tests. Thus, the changes in mechanical behavior between native murine aortas, decellularized guinea pig aortas (the grafts) and degraded aortas (AAAs) were investigated. It was shown that decellularized and degraded aortas exhibited a different mechanical behavior than native murine aortas. Indeed, decellularized aortas presented a marked decrease in circumferential stretch and distensibility compared with native aortas. Moreover, we evidenced an exacerbation of these changes in mechanical behavior for AAAs, which showed the lowest distension and distensibility at 100mmHg. The opening angle test also revealed a complete loss of residual stresses in the degraded arterial wall given the non opening of rings extracted from AAAs.

Salidroside contributes to reducing blood pressure and alleviating cerebrovascular contractile activity in diabetic Goto-Kakizaki Rats by inhibition of L-type calcium channel in smooth muscle cells

BackgroundVascular disease is a common and often severe complication in diabetes mellitus. Hyperglycemia and hypertension are considered to be two of the leading risk factors for vascular complications in diabetic patients. However, few pharmacologic agents could provide a combinational therapy for controlling hyperglycemia and blood pressure in diabetic patients at the same time. Salidroside (SAL) is the major active ingredient derived from Rhodiola. Recently, it has been reported that SAL have an obvious hypoglycemic effect in diabetes and show a beneficial activity in diabetic vascular dysfunction. However, it remains unknown whether or not SAL treatment could directly reduce blood pressure in diabetes. Furthermore, it is not clear what is the molecular mechanism underlying the vascular protection of SAL treatment in diabetes.MethodsMale diabetic Goto-Kakizaki (GK) and non-diabetic control Wistar-Kyoto (WKY) rats were administrated with different dosages of SAL (50, 100 and 200 mg/kg/day) for 4 weeks. Contractile responsiveness of cerebral artery to KCl or 5-HT was investigated by Pressure Myograph System. The activity of CaL channel was investigated by recording whole-cell currents, assessing the expressions of CaL channel α1C-subunit and its downstream kinase, MLCK, at protein or mRNA levels.ResultsWe showed that administration of 100 mg/kg/day SAL for 4 weeks not only lowered blood glucose, but also reduced blood pressure and alleviated cerebrovascular contractile activity in diabetic GK rats, which suggested that SAL treatment may provide a combinational therapy for lowering blood glucose and reducing blood pressure in diabetes at the same time. Furthermore, SAL treatment markedly inhibited the function and expression of CaL channel in cerebral VSMCs isolated from diabetic GK rats or when exposed to hyperglycemia condition, which may be the underlying mechanism responsible for the vascular protection of SAL in diabetes.ConclusionsThe present study provided evidences that SAL contributes to reducing blood pressure and alleviating cerebrovascular contractile activity in diabetic GK rats by inhibition of CaL channel in smooth muscle cells, which may provide a novel approach to treat vascular complications in diabetic patients.

Increased amplitude of inward rectifier K+ currents with advanced age in smooth muscle cells of murine superior epigastric arteries.

Inward rectifier K+ channels (KIR) may contribute to skeletal muscle blood flow regulation and adapt to advanced age. Using mouse abdominal wall superior epigastric arteries (SEAs) from either young (3-6 mo) or old (24-26 mo) male C57BL/6 mice, we investigated whether SEA smooth muscle cells (SMCs) express functional KIR channels and how aging may affect KIR function. Freshly dissected SEAs were either enzymatically dissociated to isolate SMCs for electrophysiological recording (perforated patch) and mRNA expression or used intact for pressure myography. With 5 mM extracellular K+ concentration ([K+]o), exposure of SMCs to the KIR blocker Ba2+ (100 μM) had no significant effect (P > 0.05) on whole cell currents elicited by membrane potentials spanning -120 to -30 mV. Raising [K+]o to 15 mM activated Ba2+-sensitive KIR currents between -120 and -30 mV, which were greater in SMCs from old mice than in SMCs from young mice (P < 0.05). Pressure myography of SEAs revealed that while aging decreased maximum vessel diameter by ~8% (P < 0.05), it had no significant effect on resting diameter, myogenic tone, dilation to 15 mM [K+]o, Ba2+-induced constriction in 5 mM [K+]o, or constriction induced by 15 mM [K+]o in the presence of Ba2+ (P > 0.05). Quantitative RT-PCR revealed SMC expression of KIR2.1 and KIR2.2 mRNA that was not affected by age. Barium-induced constriction of SEAs from young and old mice suggests an integral role for KIR in regulating resting membrane potential and vasomotor tone. Increased functional expression of KIR channels during advanced age may compensate for other age-related changes in SEA function.NEW & NOTEWORTHY Ion channels are integral to blood flow regulation. We found greater functional expression of inward rectifying K+ channels in smooth muscle cells of resistance arteries of mouse skeletal muscle with advanced age. This adaptation to aging may contribute to the maintenance of vasomotor tone and blood flow regulation during exercise.

Abnormal Remodeling of Subcutaneous Small Arteries Is Associated With Early Diastolic Impairment in Metabolic Syndrome

BackgroundSmall artery pathophysiology is frequently invoked as a cause of obesity‐related diastolic heart failure. However, evidence to support this hypothesis is scant, particularly in humans.Methods and ResultsTo address this, we studied human small artery structure and function in obesity and looked for correlations between vascular parameters and diastolic function. Seventeen obese patients with metabolic syndrome and 5 control participants underwent echocardiography and subcutaneous gluteal fat biopsy. Small arteries were isolated from the biopsy and pressure myography was used to study endothelial function and wall structure. In comparison with the control group, small arteries from obese participants exhibited significant endothelial dysfunction, assessed as the vasodilatory response to acetylcholine and also pathological growth of the wall. For the obese participants, multiple regression analysis revealed an association between left atrial volume and both the small artery wall thickness (β=0.718, P=0.02) and wall‐to‐lumen ratio (β=0.605, P=0.02). Furthermore, the E:E′ ratio was associated with wall‐to‐lumen ratio (β=0.596, P=0.02) and inversely associated with interleukin‐6 (β=−0.868, P=0.03). By contrast, endothelial function did not correlate with any of the echocardiographic parameters studied.ConclusionsAlthough the small arteries studied were not cardiac in origin, our results support a role for small artery remodeling in the development of diastolic dysfunction in humans. Further direct examination of the structure and function of the myocardial resistance vasculature is now warranted, to elucidate the temporal association between metabolic risk factors, small artery injury, and diastolic impairment.

Chronic Cerebral Hypoperfusion Impairs Transient Receptor Potential Channel Vanilloid 4 (TRPV4)‐Mediated Dilation in Cerebral Parenchymal Arterioles From Normotensive, but not Hypertensive Rats

Hypertension is a risk factor for cerebral small vessel disease and vascular cognitive impairment. Hypertension also increases the risk of other vascular injuries, such as carotid artery stenosis, that can place additional stress on the cerebral vasculature. To understand how hypertension affects the vascular response to reduced cerebral perfusion we studied normotensive Wistar Kyoto (WKY) rats and stroke prone spontaneously hypertensive rats (SHSRP). Rats underwent a bilateral carotid artery stenosis (BCAS) surgery or a sham surgery at 20 weeks of age, and they were studied 8 weeks later. In both cases the BCAS surgery impaired short‐term memory assessed by calculating the novel object exploration quotient (WKY: 0.6±0.04 vs 0.4±0.4, sham vs BCAS; n=11–13, p&lt;0.05; SHRSP: 0.6±0.05 vs 0.5±0.04 sham vs BCAS; n=16, p&lt;0.05). The effects of BCAS on the parenchymal arteriole (PA) function were assessed by pressure myography. BCAS had no effect of myogenic tone generation in either rat strain. In both WKY rats and SHRSP, BCAS caused a marked impairment in carbachol mediated endothelium‐dependent dilation (% dilation, WKY: 29.6±8.1 vs −10.2±7.4, sham vs BCAS; n=5–7, p&lt;0.05; SHRSP: 17.5±5.3 vs −0.22±2.9, sham vs BCAS; n=5–6 p&lt;0.05). Recent studies from our lab suggest that transient receptor potential channel vanilloid 4 (TRPV4) mediates endothelium‐dependent dilation in PAs. To assess the effects of BCAS on TRPV4‐dependent dilation, we used GSK1016790A, a potent and selective TRPV4 agonist. We found that TRPV4‐mediated dilation in the WKY rats was significantly impaired post‐BCAS (% dilation, 93.5±3.3 vs 57.7±13.3 Sham vs BCAS; n=3–4, p&lt;0.05); this effect was not observed in the SHRSP (% dilation, 57.2±1.6 vs 51.8±6.9 sham vs BCAS; n=5–6, p&lt;0.05). Interestingly TRPV4‐mediated dilation was impaired in the sham hypertensive rats compared to the sham WKY rats suggesting that hypertension impacts TRPV4 function. Together these data indicate that impaired TRPV4‐mediated dilation may contribute to the cognitive decline observed with BCAS in normotensive rats but not in rats with sustained hypertension.Support or Funding InformationSupported by 5P01HL070687‐12.

Matrix Metalloproteinase (MMP)‐2 Diverges in Regulate Calponin‐1 in Conductance and Resistance Arteries in Early Hypertension‐Induced Vascular Dysfunction and Remodeling

IntroductionIncreased activity of MMP‐2 degrades many extra‐ and intracellular proteins, an effect that may contribute to hypertension‐induced vascular remodeling and dysfunction. Calponin‐1 is a differetiation marker of vascular smooth muscle cells (VSMC) and also inhibits Mg‐ATPase activity of myosin to regulate contraction. Calponin‐1 is an intracellular proteolytic target of MMP‐2 in aortas of hypertensive rats. The aim of this study is to investigate whether MMP‐2 contributes to hypertension‐induced vascular remodeling and dysfunction of conductance and resistance arteries by regulating calponin‐1.MethodsMale wistar rats were submitted to the two kidney‐one clip (2K‐1C) model of hypertension or sham surgery and were treated with doxycycline (inhibitor of MMPs, 30 mg/kg/day) or water for one week. Systolic blood pressure (SBP) was daily accessed by tail cuff plethysmography. After one week, aortas and mesenteric arteries were removed to analyze the activity of MMP‐2 by in situ zymography and calponin‐1 by immunofluorescence. Morphological analysis was performed with hematoxylin and eosin staining in aortas and pressurized myograph in mesenteric arteries. Immunohistochemistry to Ki‐67 was performed to analyze celular proliferation. Phenylephrine (PE) concentration‐effect curve was performed in aortas and in the third branch of mesenteric arteries by using tension myography. Statistical analysis were done by two‐way ANOVA. The Ethics Committee in Animal Research approved all protocols (012/2015‐1).ResultsSBP increased in the 2K‐1C rats (145.8±1.6 vs. 118.2±2.9, p&lt;0,05) and treatment with doxycycline did not reduce it. The morphological analysis showed that 2K‐1C aortas had an increase in the media per lumen ratio (p&lt;0,05) and cross‐sectional area (p&lt;0,05) vs. Sham groups, an alteration that was not observed in mesenteric arteries at early hypertension. Furthermore, doxycycline was able to revert the increased VSMCs proliferation in aortas of 2K‐1C rats (p&lt;0,05). On the other hand, PE significantly contracted more the mesenteric arteries of 2K‐1C (p&lt;0.05 vs. Sham), and doxycycline reverted it. The potency of PE to contract aortas of 2K‐1C rats was higher than controls and doxycycline also reverted it. MMP‐2 activity was increased in both arterial bed of 2K‐1C rats and doxycycline reduced it (p&lt;0,05). While calponin‐1 was lower in the aortas of 2K‐1C rats and treatment ameliorated it (p&lt;0,05), its levels were increased in the 2K‐1C mesenteric arteries (p&lt;0,05).ConclusionIn conductance arteries, the reduction of calponin‐1 by MMP‐2 contributed to VSMC proliferation and phenotype switch, which may result in the hypertrophic remodeling of hypertension. In resistance arteries, MMP‐2 may contribute to increase calponin‐1 as a regulatory mechanism to compensate hypertension‐induced increased vascular resistance. This effect may be particularly related to the extracellular mechanisms of MMP‐2.Support or Funding InformationCNPq, CAPES and FAPESP

The pro-inflammatory cytokine TNF-α inhibits lymphatic pumping via activation of the NF-κB-iNOS signaling pathway.

Mesenteric lymphatic vessel pumping, important to propel lymph and immune cells from the intestinal interstitium to the mesenteric lymph nodes, is compromised during intestinal inflammation. The objective of this study was to test the hypothesis that the pro-inflammatory cytokine TNF-α, is a significant contributor to the inflammation-induced lymphatic contractile dysfunction, and to determine its mode of action. Contractile parameters were obtained from isolated rat mesenteric lymphatic vessels mounted on a pressure myograph after 24-hours incubation with or without TNF-α. Various inhibitors were administered, and quantitative real-time PCR, Western blotting, and immunofluorescence confocal imaging were applied to characterize the mechanisms involved in TNF-α actions. Vessel contraction frequency was significantly decreased after TNF-α treatment and could be restored by selective inhibition of NF-кB, iNOS, guanylate cyclase, and ATP-sensitive K+ channels. We further demonstrated that NF-кB inhibition also suppressed the significant increase in iNOS mRNA observed in TNF-α-treated lymphatic vessels and that TNF-α treatment favored the nuclear translocation of the p65 NF-κB subunit. These findings suggest that TNF-α decreases mesenteric lymphatic contractility by activating the NF-κB-iNOS signaling pathway. This mechanism could contribute to the alteration of lymphatic pumping reported in intestinal inflammation.

Local Delivery of β‐Aminopropionitrile Improves Arteriovenous Fistula Maturation and Remodeling

IntroductionArteriovenous fistula (AVF) is the preferred vascular access to provide hemodialysis in patients with end stage renal disease (ESRD). The failure of AVF due to improper remodeling is a serious condition that affects not only its prevalence but also the possibility of achieving long‐term patency. β‐aminopropionitrile (BAPN) is an irreversible inhibitor of lysyl oxidase (LOX), the main enzyme responsible for collagen crosslinking. Herein, we hypothesize that inhibition of LOX by local delivery of BAPN improves biomechanical performance in experimental AVF and prevents excessive crosslinking of collagen and other extracellular matrix proteins.MethodsPoly Lactic‐co‐Glycolic Acid (PLGA) is an FDA‐approved biodegradable highly biocompatible polymer frequently used on deliveries systems. The scaffolds were grown by electrospinning PLGA/BAPN (15:1.5) in hexafluoroisopropanol at 20 keV and 13 cm away from the target with a flow of 2 mL/h. In vitro degradation as well as BAPN release profile was studied during 60 days at 37°C. BAPN concentration was assessed with a Triversa NanoMate® electrospray ionization with direct infusion through triple quadrupole LC‐MS. Two groups of Sprague Dawley rats of both sex received local delivery of scaffold containing BAPN (0.27 mg) or vehicle around the venous limb of the newly created AVF. The rat AVF was created by surgically anastomosing the left epigastric vein to the nearby femoral artery. Pressure myograph (110 P; Danish Myo Technology, Aarhus, Denmark) was used for assessment of the AVF mechanical properties.ResultsIn‐vitro release assessment showed that the drug inclusion in the scaffolds affects degradation rate with a maximum difference of 70% at 40 days. LC‐MS showed an excellent sensitivity for the quantification of BAPN with a lower limit of quantification of 30 ng/mL. Both treated and control AVF increased its size more than double that of the contralateral epigastric vein (p&lt;0.05). However, local delivery of BAPN increased lumen volume in treated AVF with respect to control vessels (p=0.0078), Figure 1.ConclusionOur results demonstrate that inhibition of collagen cross‐linking by BAPN significantly improved vascular compliance and biomechanical properties of AVF.Support or Funding InformationThis research it is support as a Research Supplements to Promote Diversity in Health under parent award R01DK098511.

Purinergic signalling in mesenteric vascular smooth muscle in a mice model of essential hypertension

In vascular smooth muscle cells (VSMCs) non‐selective cation channels such as TRPC3 and TRPC6 have been proposed as the molecular constituents of receptor‐(ROCs) and stretch‐(SOCs) operated channels linking DAG signaling cascade pathway with increased vascular tone. The membrane depolarization caused by TRPCs can increase Ca2+ entry leading to augmented [Ca2+]I and VSMC contraction. Therefore, by influencing membrane potential, TRPC channels could play an important role controlling the increased vascular tone present in several pathologies, such as essential hypertension. We have previously described an increased functional expression of TRPC3 channels in mesenteric VSMCs from hypertensive mice that can contribute to increased vascular tone by favouring depolarization (Alvarez‐Miguel et al., J Physiol, in press). As several mechanosensitive G‐protein coupled receptors (GPCR) are linked to the DAG‐dependent activation of TRPC3/6 channels, we explore here the contribution of this signalling pathway to the increased vascular tone in hypertension.Using a mouse model of essential hypertension (BPH mice) and its normotensive control (BPN mice) we explored changes in the molecular components of the mechanotransduction pathway in hypertension. We analized the expression of mechanosensitive ion channels and some VSMCs sensor elements, including GPCRs, channels and transporters with qPCR. Their functional contribution was studied in isolated VSMCs (using electrophysiology) and in intact mesenteric arteries with pressure myography.Real‐time PCR showed an increased expression of some of the elements of the purinergic signalling pathway, including purinergic receptors P2Y2 and P2Y6, TRPC3 channels and the Ca2+‐activated Cl− channel Clca1. Pressure myography of mesenteric arteries after endothelium removal showed a vasoconstriction response to ATP, UTP and UDP. ATP‐induced vasoconstriction was not different between BPN and BPH arteries. The EC50 of the effect was around 7–10 μM in both preparations, and it could be blocked by α‐β‐methilene‐ATP. UTP‐induced vasoconstriction was not affected by α‐β‐methilene‐ATP and was significantly larger in BPH arteries: 50 μM UTP‐elicited vasoconstriction represented 40% of the maximal response to ATP in BPN arteries and 150% in BPH arteries. Our data indicate that mesenteric vascular reactivity to ATP is not altered in our hypertension model, but there is an increased contribution of the purinergic GPCR signalling pathway to the increased vascular tone. Together with the observed upregulation of P2YR receptors in BPH VSMCs, the augmented functional expression of TRPC3 channels could participate in the increased vasoconstriction observed in response to uridine nucleotides in BPH mesenteric arterieSupport or Funding InformationSupported by grants ISCIII‐FEDER RD12/0042/0006, (Heracles Program), BFU2013‐45867‐R (MINECO) and by a Junta de Castilla y Leon fellowship to IAM

Novel RAGE Antagonist, FPS‐ZM1, Reverses Mesenteric Arteriolar Remodeling in Type 2 Diabetic db/db Mice.

Type II diabetes (T2D) is a major risk factor for cardiovascular diseases. Recently, our laboratory established a differential effect of T2D on vascular remodeling that was dependent upon vessel size and location. In addition, our preliminary data showed an increase in circulating levels of advanced glycation end products (AGE) in T2D mice. Activation of AGE receptors (RAGE) was shown to be associated with vascular dysfunction and vascular smooth muscle proliferation, which may result in vascular remodeling. In the present study, we hypothesized that pharmacological RAGE blockade using a novel antagonist would reverse microvascular remodeling observed in T2D. Using a pressure myograph system, passive vascular mechanics were assessed in aortae and mesenteric arterioles (MA) isolated from 16 week‐old lean control mice (Db/db), obese T2D mice (db/db) and T2D mice (db/db+FPS) treated with RAGE antagonist (FPS‐ZM1, 1mg/Kg/day ip, 28 days). Body weight (BW) and fasting blood glucose (BG) were significantly increased in diabetic db/db compared to control Db/db mice (BW: 29.8±0.5 in Db/db vs. 47.3± 1.5 g db/db, p&lt;0.05 and BG: 185 Db/db ± 13 vs 687 ± 20 mg/dL in db/db, p&lt;0.05). FPS‐ZM‐1 treatment did not affect BW (46.3±1.5g) nor BG (679±17 mg/dL) in T2D mice. In aorta, neither diabetes nor RAGE antagonism in diabetic mice affected the macrovascular mechanical properties. In contrast, MAs isolated from T2D mice exhibited a significant increase in internal diameter (158±2 in Db/db vs. 208±13 μm in db/db at 100mmHg, p&lt;0.05), external diameter (180±3 in Db/db vs. 233±14 μm in db/db at 100mmHg, p&lt;0.05) and cross‐sectional area (CSA) (6066±597 in Db/db vs. 8753±881 μm2 in db/db at 100mmHg, p&lt;0.05). RAGE antagonism in T2D significantly reduced MAs internal diameter (178±5 in db/db+FPS vs. 208±13 μm in db/db at 100mmHg, p&lt;0.05), external diameter (200±5 in db/db+FPS vs. 233±14 μm in db/db at 100mmHg, p&lt;0.05) and CSA (6453±400 in db/db+FPS vs. 8753±881 μm2 in db/db at 100mmHg, p&lt;0.05). Together, our data showed a variation in macrovascular vs. microvascular remodeling in T2D as we have previously shown. Furthermore, our data suggest that AGE/RAGE signaling contributes to the microvascular remodeling observed in T2D and that RAGE blockade using a novel antagonist may present a potential therapeutic target to reduce or reverse diabetes‐mediated vascular complications.Support or Funding InformationR00 HL116769 and Nationwide Children's Hospital

Microvascular Endothelial Dysfunction in Obesity Is Driven by Macrophage-Dependent Hydrogen Sulfide Depletion.

The function of perivascular adipose tissue as an anticontractile mediator in the microvasculature is lost during obesity. Obesity results in inflammation and recruitment of proinflammatory macrophages to the perivascular adipose tissue that is paralleled by depletion of the vasorelaxant signaling molecule hydrogen sulfide (H2S) in the vessel. The current objective was to assess the role of macrophages in determining vascular [H2S] and defining how this impinged on vasodilation. Contractility and [H2S] were measured in mesenteric resistance arterioles from lean and obese mice by using pressure myography and confocal microscopy, respectively. Vasodilation was impaired and smooth muscle and endothelial [H2S] decreased in vessels from obese mice compared with those from lean controls. Coculturing vessels from lean mice with macrophages from obese mice, or macrophage-conditioned media, recapitulated obese phenotypes in vessels. These effects were mediated by low molecular weight species and dependent on macrophage inducible nitric oxide synthase activity. The inducible nitric oxide synthase activity of perivascular adipose tissue-resident proinflammatory macrophages promotes microvascular endothelial dysfunction by reducing the bioavailability of H2S in the vessel. These findings support a model in which vascular H2S depletion underpins the loss of perivascular adipose tissue anticontractile function in obesity.