Human Resistance Arteries Research Articles

Impairment of microvascular endothelial Kir2.1 channels contributes to endothelial dysfunction in human hypertension.

Hypertension is associated with decreased endothelial function through reduced contributions of nitric oxide (NO). We previously discovered that flow-induced NO production in resistance arteries of mice and humans critically depends on endothelial inwardly rectifying K+ (Kir2.1) channels. The goal of this study was to establish whether these channels contribute to the impairment of endothelial function, measured by flow-induced vasodilation (FIV) in peripheral resistance arteries of humans with hypertension. We measured FIV in vessels isolated from subcutaneous fat biopsies from 32 subjects: normotensive [n = 19; 30.6 ± 9.8 yr old; systolic blood pressure (SBP): 115.2 ± 7 mmHg; diastolic blood pressure (DBP): 75.3 ± 5.7 mmHg] and hypertensive (n = 13; 45.3 ± 15.3 yr old; SBP: 146.1 ± 15.2 mmHg; DBP: 94.4 ± 6.9 mmHg). Consistent with previous studies, we find that FIV is impaired in hypertensive adults as demonstrated by a significant reduction in FIV when compared with the normotensive adults. Furthermore, our data suggest that the impairment of FIV in hypertensive adults is partially attributed to a reduction in Kir2.1-dependent vasodilation. Specifically, we show that blocking Kir2.1 with ML133 or functionally downregulating Kir2.1 with endothelial-specific adenoviral vector containing dominant-negative Kir2.1 (dnKir2.1) result in a significant reduction in FIV in normotensive subjects but with a smaller effect in hypertensive adults. The Kir2.1-dependent vasodilation was negatively correlated to both SBP and DBP, indicating that the Kir2.1 contribution to FIV decreases as blood pressure increases. In addition, we show that exposing vessels from normotensive adults to acute high-pressure results in loss of Kir2.1 contribution, as high pressure impairs vasodilation. No effect is seen when these vessels were incubated with dnKir2.1. Overexpressing wtKir2.1 in the endothelium resulted in some improvement in vasodilation in arteries from all participants, with a greater recovery in hypertensive adults. Our data suggest that hypertension-induced suppression of Kir2.1 is an important mechanism underlying endothelial dysfunction in hypertension.NEW & NOTEWORTHY Impairment of endothelial function under high blood pressure is linked to the loss of inwardly rectifying K+ (Kir2.1) channels activity in human resistance arteries, leading to a reduction in flow-induced vasodilation and possibly leading to a vicious cycle between elevation of blood pressure, and further impairment of Kir2.1 function and flow-induced vasodilation.

PKC Inhibition Improves Human Penile Vascular Function and the NO/cGMP Pathway in Diabetic Erectile Dysfunction: The Role of NADPH Oxidase.

Erectile dysfunction (ED) is a frequent and difficult-to-treat condition in diabetic men. Protein kinase C (PKC) is involved in diabetes-related vascular and cavernosal alterations. We aimed to evaluate the role of PKC in endothelial dysfunction and NO/cGMP impairment associated with diabetic ED in the human corpus cavernosum (CC) and penile resistance arteries (PRAs) and the potential mechanisms involved. Functional responses were determined in the CC and PRAs in patients with non-diabetic ED and diabetic ED undergoing penile prosthesis insertion. PKC activator 12,13-phorbol-dibutyrate (PDBu) impaired endothelial relaxations and cGMP generation in response to acetylcholine in the CC from non-diabetic ED. PDBu also impaired responses to a PDE5 inhibitor, sildenafil, in non-diabetic ED patients. Conversely, a PKC inhibitor, GF109203X, improved endothelial, neurogenic, and PDE5-inhibitor-induced relaxations and cGMP generation only in the CC in diabetic ED patients. Endothelial and PDE5-inhibitor-induced vasodilations of PRAs were potentiated only in diabetes. Improvements in endothelial function in diabetes were also achieved with a specific inhibitor of the PKCβ2 isoform or an NADPH-oxidase inhibitor, apocynin, which prevented PDBu-induced impairment in non-diabetic patients. PKC inhibition counteracted NO/cGMP impairment and endothelial dysfunction in diabetes-related ED, potentially improving response to PDE5 inhibition.

Increased stiffness of omental arteries from late pregnant women at advanced maternal age.

Advanced maternal age (≥35 years) is a risk factor for poor pregnancy outcomes. Pregnancy requires extensive maternal vascular adaptations, and with age, our blood vessels become stiffer and change in structure (collagen and elastin). However, the effect of advanced maternal age on the structure of human resistance arteries during pregnancy is unknown. As omental resistance arteries contribute to blood pressure regulation, assessing their structure in pregnancy may inform on the causal mechanisms underlying pregnancy complications in women of advanced maternal age. Omental fat biopsies were obtained from younger (<35 years) or advanced maternal age (≥35 years) women during caesarean delivery (n = 7-9/group). Arteries (200-300 µm) were isolated and passive mechanical properties (circumferential stress and strain) assessed with pressure myography. Collagen (Masson's Trichrome) and elastin (Verhoff) were visualized histologically and % positively-stained area was assessed. Median maternal age was 32 years (range 25-34) for younger, and 38 years (range 35-42) for women of advanced maternal age. Circumferential strain was lower in arteries from advanced maternal age versus younger women but circumferential stress was not different. Omental artery collagen levels were similar, while elastin levels were lower with advanced maternal age versus younger pregnancies. The collagen:elastin ratio was greater in arteries from advanced maternal age versus younger women. In conclusion, omental arteries from women of advanced maternal age were less compliant with less elastin compared with arteries of younger controls, which may affect how vascular stressors are tolerated during pregnancy. Understanding how vascular aging affects pregnancy adaptations may contribute to better pregnancy outcomes.

Endothelial beta-globin is differentially expressed across mammalian species

Globin proteins interact with endothelial nitric oxide synthase at the myoendothelial junctions of resistance arteries to regulate nitric oxide signaling between endothelium and smooth muscle. Human resistance arteries express both alpha and beta globin to form heterotetrameric hemoglobin (α2β2). To better understand the role of hemoglobin in human resistance arteries, we need to identify an animal model in which both alpha and beta globin are expressed in the resistance vasculature. To identify potential candidates, we used microscopy to survey a range of mammalian laboratory animals for hemoglobin in the artery wall. We isolated arteries (100-200 μm in diameter) from mesenteric tissue collected from human patients undergoing clinically-indicated surgery and from the following laboratory animals: mouse ( Mus musculus, C57BL/6 strain), rat ( Rattus norvegicus, CD strain), African thicket rat ( Grammomys surdaster), guinea pig ( Cavia porcellus), New Zealand white rabbit ( Oryctolagus cuniculus), Beagle dog ( Canis lupus familiaris), and cynomolgus macaque ( Macaca fascicularis) after they underwent euthanasia on approved animal study protocols. We leveraged the autofluorescence of heterotetrameric hemoglobin under multiphoton excitation to visualize hemoglobin in the wall of intact, unstained, paraformaldehyde-fixed artery segments. Autofluorescent puncta representing tetrameric hemoglobin were observed in the wall of resistance arteries from the human patients, non-human primate, and canines, but not from rabbit nor any of the rodent species. The ability of beta globin to restrict endothelial NO signaling was confirmed in canine arteries ex vivo by testing a beta globin mimetic peptide that inhibited vasoconstriction to phenylephrine. In conclusion, primates and canines appear to have tetrameric hemoglobin in the resistance artery wall, whereas rodents and rabbits do not. This study highlights differences in vascular globin expression between humans and rodents and extends the recent discovery of hemoglobin in resistance arteries of humans to two additional mammalian species that may serve as models for studying the unique activities of tetrameric hemoglobin in the resistance vasculature. Funding: Intramural Research program, NIAID, NIH This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Selective Partial Agonism of Vasopressin 1a Receptors In Vitro by OCE-205

Objective To test the selectivity and degree of functional agonism of Ocelot Bio’s dual agonist/antagonist molecule, OCE-205, at the vasopressin 1a receptor (V1aR). Methods Cells expressing human (h) or rat V1a, V1b, V2, or oxytocin receptors (OTR) were incubated with varying concentrations of OCE-205 or with arginine vasopressin (AVP), and responses were measured with fluorescence or reporter gene assays. In addition, human resistance arteries were exposed to increasing concentrations of OCE-205, and the resulting contractility was measured. Results The mean efficacy of OCE-205 at hV1aR was 39% of the maximal possible effect (MPE), with a mean EC50 of 0.71 nM. Above 1 nM OCE-205, the percent maximal possible effect (%MPE) plateaued. The EC50 was much higher at hV1bR (134 nM), hV2R (420 nM), and OTR (6.9 nM), indicating selectivity for hV1aR. Results at rat receptors were similar. OCE-205 produced 40.0% of maximal depolarization-induced contraction, demonstrating functional partial agonism. Conclusion The dual agonist/antagonist structure of OCE-205 thus allows it to act as a highly selective partial agonist at vasopressin V1aR at therapeutically relevant concentrations.

Vascular Smooth Muscle TRPV4 (Transient Receptor Potential Vanilloid Family Member 4) Channels Regulate Vasoconstriction and Blood Pressure in Obesity.

Vascular endothelium and smooth muscle work together to keep the balance of vasomotor tone and jointly maintain vascular homeostasis. Ca2+-permeable ion channel TRPV4 (transient receptor potential vanilloid family member 4) in endothelial cells regulates endothelium-dependent vasodilation and contraction in various states. However, how vascular smooth muscle cell TRPV4 (TRPV4SMC) contributes to vascular function and blood pressure regulation in physiological and pathologically obese condition has not been fully studied. We generated smooth muscle TRPV4-deficient mice and developed diet-induced obese mice model and analyzed the role of TRPV4SMC in intracellular Ca2+ ([Ca2+]i) regulation and vasoconstriction. Vasomotor changes of mouse mesenteric artery were measured by wire, and pressure myography. [Ca2+]i were measured by fluo-4 staining. Blood pressure was recorded by telemetric device. Vascular TRPV4SMC played different roles in regulating vasomotor tone than endothelial TRPV4 due to their different features of [Ca2+]i regulation. Loss of TRPV4SMC attenuated U46619- and phenylephrine-induced contraction, suggesting its involvement in regulating vascular contractility. Mesenteric arteries from obese mice showed SMC hyperplasia, suggesting an increased level of TRPV4SMC. Loss of TRPV4SMC did not influence the development of obesity but protected mice from obesity-induced vasoconstriction and hypertension. In arteries deficient in SMC TRPV4, SMCs F-actin polymerization and RhoA dephosphorylation were attenuated under contractile stimuli. Moreover, SMC-dependent vasoconstriction was inhibited in human resistance arteries with TRPV4 inhibitor application. Our data identify TRPV4SMC as a regulator of vascular contraction in both physiological states and pathologically obese mice. TRPV4SMC contributes to the ontogeny of vasoconstriction and hypertension induced by TRPV4SMC over-expression in obese mice mesenteric artery.

Indoxyl Sulphate Retention Is Associated with Microvascular Endothelial Dysfunction after Kidney Transplantation.

Kidney transplantation (KTx) is the preferred form of renal replacement therapy in chronic kidney disease (CKD) patients, owing to increased quality of life and reduced mortality when compared to chronic dialysis. Risk of cardiovascular disease is reduced after KTx; however, it is still a leading cause of death in this patient population. Thus, we aimed to investigate whether functional properties of the vasculature differed two years post-KTx (postKTx) compared to baseline (time of KTx). Using the EndoPAT device in 27 CKD patients undergoing living-donor KTx, we found that vessel stiffness significantly improved while endothelial function worsened postKTx vs. baseline. Furthermore, baseline serum indoxyl sulphate (IS), but not p-cresyl sulphate, was independently negatively associated with reactive hyperemia index, a marker of endothelial function, and independently positively associated with P-selectin postKTx. Finally, to better understand the functional effects of IS in vessels, we incubated human resistance arteries with IS overnight and performed wire myography experiments ex vivo. IS-incubated arteries showed reduced bradykinin-mediated endothelium-dependent relaxation compared to controls via reduced nitric oxide (NO) contribution. Endothelium-independent relaxation in response to NO donor sodium nitroprusside was similar between IS and control groups. Together, our data suggest that IS promotes worsened endothelial dysfunction postKTx, which may contribute to the sustained CVD risk.

STIM/Orai Inhibition as a Strategy for Alleviating Diabetic Erectile Dysfunction Through Modulation of Rat and Human Penile Tissue Contractility and in vivo Potentiation of Erectile Responses

Stromal interaction molecule (STIM)/Orai calcium entry system appears to have a role in erectile dysfunction (ED) pathophysiology but its specific contribution to diabetic ED was not elucidated. To evaluate STIM/Orai inhibition on functional alterations associated with diabetic ED in rat and human penile tissues and on in vivo erectile responses in diabetic rats. Rat corpus cavernosum (RCC) strips from nondiabetic (No DM) and streptozotocin-induced diabetic (DM) rats and human penile resistance arteries (HPRA) and corpus cavernosum (HCC) from ED patients undergoing penile prosthesis insertion were functionally evaluated in organ chambers and wire myographs. Erectile function in vivo in rats was assessed by intracavernosal pressure (ICP) responses to cavernous nerve electrical stimulation (CNES). Expression of STIM/Orai elements in HCC was determined by immunofluorescence and immunoblot. Functional responses in RCC, HCC and HPRA and STIM/Orai protein expression in HCC. In vivo erectile responses to CNES. Inhibition of Orai channels with YM-58483 (20 µM) significantly reduced adrenergic contractions in RCC but more effectively in DM. Thromboxane-induced and neurogenic contractions were reduced by STIM/Orai inhibition while defective endothelial, neurogenic and PDE5 inhibitor-induced relaxations were enhanced by YM-58483 (10 µM) in RCC from DM rats. In vivo, YM-58483 caused erections and attenuated diabetes-related impairment of erectile responses. YM-58483 potentiated the effects of PDE5 inhibition. In human tissues, STIM/Orai inhibition depressed adrenergic and thromboxane-induced contractions in ED patients more effectively in those with type 2 diabetes. Diabetes was associated with increased expression of Orai1 and Orai3 in ED patients. Targeting STIM/Orai to alleviate diabetes-related functional alterations of penile vascular tissue could improve erectile function and potentiate therapeutic effects of PDE5 inhibitors in diabetic ED. Improving effects of STIM/Orai inhibition on diabetes-related functional impairment was evidenced in vitro and in vivo in an animal model and validated in human tissues from ED patients. Functional findings were complemented with expression results. Main limitation was low numbers of human experiments due to limited human tissue availability. STIM/Orai inhibition alleviated alterations of functional responses in vitro and improved erectile responses in vivo in diabetic rats, potentiating the effects of PDE5 inhibition. STIM/Orai inhibition was validated as a target to modulate functional alterations of human penile vascular tissue in diabetic ED where Orai1 and Orai3 channels were upregulated. STIM/Orai inhibition could be a potential therapeutic strategy to overcome poor response to conventional ED therapy in diabetic patients. Sevilleja-Ortiz A, El Assar M, García-Gómez B, et al. STIM/Orai Inhibition as a Strategy for Alleviating Diabetic Erectile Dysfunction Through Modulation of Rat and Human Penile Tissue Contractility and in vivo Potentiation of Erectile Responses. J Sex Med 2022;19:1733-1749.

Alpha globin gene copy number and hypertension risk among Black Americans.

BackgroundAlpha globin is expressed in the endothelial cells of human resistance arteries where it binds to endothelial nitric oxide synthase and limits release of the vasodilator nitric oxide. Genomic deletion of the alpha globin gene (HBA) is common among Black Americans and could lead to increased endothelial nitric oxide signaling and reduced risk of hypertension.MethodsCommunity-dwelling US adults aged 45 years or older were enrolled and examined from 2003 to 2007, followed by telephone every 6 months, and reexamined from 2013 to 2016. At both visits, trained personnel performed standardized, in-home blood pressure measurements and pill bottle review. Prevalent hypertension was defined as systolic blood pressure ≥ 140mmHg or diastolic blood pressure ≥ 90mmHg or anti-hypertensive medication use. Droplet digital PCR was used to determine HBA copy number. The associations of HBA copy number with prevalent hypertension, resistant hypertension, and incident hypertension were estimated using multivariable regression.ResultsAmong 9,684 Black participants, 7,439 (77%) had hypertension at baseline and 1,044 of those had treatment-resistant hypertension. 1,000 participants were not hypertensive at baseline and participated in a follow up visit; 517 (52%) developed hypertension over median 9.2 years follow-up. Increased HBA copy number was not associated with prevalent hypertension (PR = 1.00; 95%CI 0.98,1.02), resistant hypertension (PR = 0.95; 95%CI 0.86,1.05), or incident hypertension (RR = 0.96; 95%CI 0.86,1.07).ConclusionsThere were no associations between increased HBA copy number and risk of hypertension. These findings suggest that variation in alpha globin gene copy number does not modify the risk of hypertension among Black American adults.

DPP4 Promotes Human Endothelial Cell Senescence and Dysfunction via the PAR2-COX-2-TP Axis and NLRP3 Inflammasome Activation.

Abnormal accumulation of senescent cells in the vessel wall leads to a compromised vascular function contributing to vascular aging. Soluble DPP4 (dipeptidyl peptidase 4; sDPP4) secretion from visceral adipose tissue is enhanced in obesity, now considered a progeric condition. sDPP4 triggers vascular deleterious effects, albeit its contribution to vascular aging is unknown. We aimed to explore sDPP4 involvement in vascular aging, unraveling the molecular pathway by which sDPP4 acts on the endothelium. Human endothelial cell senescence was assessed by senescence-associated β-galactosidase assay, visualization of DNA damage, and expression of prosenescent markers, whereas vascular function was evaluated by myography over human dissected microvessels. In visceral adipose tissue biopsies from a cohort of obese patients, we explored several age-related parameters in vitro and ex vivo. By a common mechanism, sDPP4 triggers endothelial cell senescence and endothelial dysfunction in isolated human resistance arteries. sDPP4 activates the metabotropic receptor PAR2 (protease-activated receptor 2), COX-2 (cyclooxygenase 2) activity, and the production of TXA2 (thromboxane A2) acting over TP (thromboxane receptor) receptors (PAR2-COX-2-TP axis), leading to NLRP3 (nucleotide-binding oligomerization domain, leucine-rich repeat, and pyrin domain-containing 3) inflammasome activation. Obese patients exhibited impaired microarterial functionality in comparison to control nonobese counterparts. Importantly, endothelial dysfunction in obese patients positively correlated with greater expression of DPP4, prosenescent, and proinflammatory markers in visceral adipose tissue nearby the resistance arteries. Moreover, when DPP4 activity or sDPP4-induced prosenescent mechanism was blocked, endothelial dysfunction was restored back to levels of healthy subjects. These results reveal sDPP4 as a relevant mediator in early vascular aging and highlight its capacity activating main proinflammatory mediators in the endothelium that might be pharmacologically tackled.

Cardiovascular and hemodynamic consequences of recombinant placental growth factor administration in Guinea pigs

ABSTRACT Placental growth factor (PlGF), a member of the vascular endothelial growth factor family of proteins regulating angiogenesis, has been shown to have acute vasodilatory effects on human resistance arteries. However, the acute hemodynamic effects of PlGF in vivo are not known. The aim of this study was to report acute cardiovascular changes induced by recombinant human PlGF administered intravenously in male Guinea Pigs with implanted telemeters. PlGF decreased mean arterial blood pressure by 10–20% within minutes. The magnitude of reduction was similar at three dose levels; however, the duration of relative hypotension was dose-dependent. Blood pressure reduction resulted in a compensatory increase in heart rate, or reflex tachycardia. To rule out any direct effect on the heart, PlGF was tested in the ex vivo Langendorff heart preparation, and no cardiac changes were found. Together these results suggest that the PlGF-related changes in blood pressure are largely mediated by its actions in the vasculature.

Early manifestation of aging-related vascular dysfunction in human penile vasculature-A potential explanation for the role of erectile dysfunction as a harbinger of systemic vascular disease.

Advanced age is related to functional alterations of human vasculature, but erectile dysfunction precedes systemic manifestations of vascular disease. The current study aimed to simultaneously evaluate the influence of aging on vascular function (relaxation and contraction responses) in systemic human vascular territories: aorta (HA) and resistance mesenteric arteries (HMA) and human corpus cavernosum (HCC) and penile resistance arteries (HPRA). Associations of oxidative stress and inflammation circulating biomarkers with age and functional responses were also determined. Vascular specimens were obtained from 76 organ donors (age range 18-87). Four age-groups were established: < 40, 40-55, 56-65 and > 65years old. Increasing age was associated with a decline in endothelium-dependent relaxation induced by BK in HMA (r = -0.597, p = 0.0001), or by ACh in HCC (r = -0.505, p = 0.0022), and HPRA (r = -0.601, p = 0.0012). Significant impairment was detected at > 65years old in HMA but earlier in penile vasculature (> 55years old). Age-related reduction to H2O2-vasodilatory response started before in HCC (56-65years old) than in HA (> 65years old). In contrast to relaxation responses, aging-related hypercontractility to adrenergic stimulation was homogeneous: contractions significantly increased in subjects > 55years old in all tested vessels. Although not significantly age related, circulating levels of ADMA (r = -0.681, p = 0.0052) and TNF-α (r = -0.537, p = 0.0385) were negatively correlated with endothelial vasodilation in HMA but not in HCC or HPRA. Penile vasculature exhibits an early impairment of endothelium-dependent and H2O2-induced vasodilations when compared to mesenteric microcirculation and aorta. Therefore, functional susceptibility of penile vasculature to the aging process may account for anticipation of erectile dysfunction to systemic manifestations of vascular disease.

Ageing-induced hypercontractility is related to functional enhancement of STIM/Orai and upregulation of Orai 3 in rat and human penile tissue

The role of STIM/Orai calcium entry system on vascular ageing has not been elucidated. We aimed to evaluate the influence of ageing on STIM/Orai signalling and its role on ageing-induced alterations of contractile function in rat corpus cavernosum (RCC) and human penile resistance arteries (HPRA) and corpus cavernosum (HCC). RCC was obtained from 3 months-old and 20 months-old animals. HPRA and HCC were obtained from organ donors of varied ages without history of erectile dysfunction. Aging was associated with enhanced norepinephrine (NE)- and thromboxane analogue (U46619)-induced contractions in RCC which were significantly inhibited by the STIM/Orai inhibitor, YM-58483 (20 μM). Other STIM/Orai inhibitor, 2-aminoethyldiphenylborate also reduced NE-induced contractions in RCC from aged rats. YM-58483 significantly reduced neurogenic contractions and potentiated neurogenic relaxations in RCC from aged rats. In HCC and HPRA, NE-induced contractions were significantly enhanced in older subjects (>65 years-old) but YM-58483 completely reversed ageing-related hypercontractility. Ageing did not modify STIM-1 and Orai1 protein expressions but Orai3 was significantly overexpressed in cavernosal tissue from old rats and older subjects. Contribution of STIM/Orai to cavernosal contraction increases with ageing together with increased expression of Orai3. Orai inhibition could be a potential therapeutic strategy to reduce ageing-related impact on vascular/erectile function.

Microstructural Characterization of Resistance Artery Remodelling in Diabetes Mellitus

Introduction: Microvascular remodelling is a symptom of cardiovascular disease. Despite the mechanical environment being recognized as a major contributor to the remodelling process, it is currently only understood in a rudimentary way. Objective: A morphological and mechanical evaluation of the resistance vasculature in health and diabetes mellitus. Methods: The cells and extracellular matrix of human subcutaneous resistance arteries from abdominal fat biopsies were imaged using two-photon fluorescence and second harmonic generation at varying transmural pressure. The results informed a two-layer mechanical model. Results: Diabetic resistance arteries reduced in wall area as pressure was increased. This was attributed to the presence of thick, straight collagen fibre bundles that braced the outer wall. The abnormal mechanical environment caused the internal elastic lamina and endothelial and vascular smooth muscle cell arrangements to twist. Conclusions: Our results suggest diabetic microvascular remodelling is likely to be stress-driven, comprising at least 2 stages: (1) Laying down of adventitial bracing fibres that limit outward distension, and (2) Deposition of additional collagen in the media, likely due to the significantly altered mechanical environment. This work represents a step towards elucidating the local stress environment of cells, which is crucial to build accurate models of mechanotransduction in disease.

Pharmacological, Pharmacokinetic, Pharmacodynamic and Physicochemical Characterization of FE 205030: A Potent, Fast Acting, Injectable CGRP Receptor Antagonist for the Treatment of Acute Episodic Migraine

The neuropeptide calcitonin gene-related peptide (CGRP) is known to play a central role in the underlying pathophysiology of migraine. In comparison to the effective triptan class of antimigraine treatments, the CGRP antagonists possess a comparable efficacy but a superior cardiovascular safety profile in patients. This paper describes the development of selective and potent peptidic CGRP antagonist, FE 205030, that has a fast onset of action and an optimal half-life (subcutaneous Tmax ~ 60 min, and t1/2 ~ 4.4 h in 80 kg pigs, respectively), which is key to prevention of the progression of debilitating migraine symptoms. The in vivo efficacy of this agent has been established a translational pharmacodynamic model (inhibition of capsaicin-induced increase in skin blood flow) in cynomolgus monkeys and shows maximal inhibitory activity at circulating concentrations of 30-100 nM. Antagonist activity of FE 205030 was characterized on CGRP-induced vasodilation in isolated human mesenteric resistance arteries in an ex vivo isometric myograph study, and FE 205030 effectively blocked CGRP-induced vasodilation with a pA2 of 9.3 ± 0.1, mean ± standard error. Multispecies allometric scaling and modeling of subcutaneous (SC) effective concentrations indicates that a dose of 10–30 mg/day is sufficient to achieve a drug exposure/target coverage of 8h, which is useful to prevent migraine recurrence in patients. The molecule also possesses appropriate physicochemical properties that allows for a convenient dosing form factor of 1 ml injection volume with a sufficient solubility and acceptable short-term stability, optimal for treatment of acute migraine episodes in patients. Hence, FE 205030 may provide an important fast-acting injectable option for patients suffering from frequent acute migraine episodes, complementary to preventative monoclonal antibodies and oral small molecule CGRP-R antagonist therapies.

The Alpha Subunit of Hemoglobin Interacts with Endothelial Nitric Oxide Synthase to Regulate Feedback Vasodilation in Human Resistance Arteries

Alpha globin, but not beta globin, is expressed in mouse resistance arteries where it binds endothelial nitric oxide (NO) synthase (eNOS) and limits NO signaling. To understand this signaling pathway in humans, we studied omental resistance arteries. Omental tissue was collected during clinically indicated surgery on protocol 13-C-0176 and transferred to the lab on ice. Human resistance arteries 80 to 200 um in diameter were microdissected, cannulated, and perfused with cold PBS to expel blood. We measured gene expression of HBA1 (2658 ± 1190 gene transcripts per 1 ng mRNA ± SEM), HBA2 (3319 ± 1667), HBB (1831 ± 725), and NOS3 (408 ± 49) using RT-ddPCR in human arteries obtained from 7 different subjects. Expression of SLC4A1, a control for blood contamination, was nearly undetectable at (4.2 ± 2.3). To determine the binding partners of vascular alpha globin, protein lysates from omental arteries from three donors were subjected to co-immunoprecipitation with an anti-alpha globin antibody. Western blot revealed both eNOS and beta globin to co-immunoprecipitate (co-IP) with alpha globin. Multiphoton imaging of perfused omental arteries revealed co-localization of alpha globin, beta globin and eNOS to distinct punctates within the plane of the internal elastic lamina (IEL). Given the broadly autofluorescent nature of these complexes (similar to hemoglobin in red blood cells), we used fluorescence lifetime imaging microscopy (FLIM) to identify distinct peak lifetimes of fluorophores labeling antibodies against each of alpha globin, beta globin, and eNOS in the complex. To interrogate the function of endothelial hemoglobin, we performed ex vivo pressure myography on omental arteries obtained from five donors. Maximal vasoconstriction of omental arteries to the alpha-1-adrenergic agonist phenylephrine (PE) was 40.8 ± 3.1% of baseline diameter. Incubation with an alpha globin mimetic peptide that disrupts binding of alpha globin to eNOS, reduced maximal constriction in response to PE to 65.9 ± 2.7% of baseline (p < 0.0001 compared to PE alone), whereas a control peptide had little effect (46.5 ± 7.6% of baseline). Inhibition of eNOS by L-NAME in mimetic-peptide treated arteries restored PE-induced vasoconstriction (44.2 ± 2.3% of baseline). We identified expression of not only alpha but also beta globin in perfused human omental arteries. Co-IP and multiphoton microscopy identified alpha globin, beta globin, and eNOS as components of a complex that localizes near the IEL, likely in myoendothelial junctions. Disruption of alpha globin-eNOS binding enhanced feedback vasodilation to phenylephrine in an NO synthase-dependent fashion. In conclusion, we report that hemoglobin is expressed in human omental arteries where it binds eNOS and regulates alpha-1-adrenergic feedback vasodilation by limiting nitric oxide signaling.

Kynurenine Relaxes Arteries of Normotensive Women and Those With Preeclampsia.

[Figure: see text].

Endothelin-1 potentiates TRPV1-mediated vasoconstriction of human adipose arterioles in a protein kinase C-dependent manner.

The TRPV cation channels have emerged as important regulators of vascular tone. TRPV1 channels and endothelin-1 are independently associated with the pathophysiology of coronary vasospasm, but the relationship between their vasomotor functions remains unclear. We characterized the vasomotor function of TRPV1 channels in human arterioles and investigated regulation of their vasomotor function by endothelin-1. Human arterioles (mainly from adipose tissue) were threaded on two metal wires, equilibrated in a physiological buffer at 37°C and exposed to increasing concentrations of capsaicin, with or without SB366791 (TRPV1-selective inhibitor) or GF109203X (PKC-selective inhibitor). Some arterioles were pre-constricted with endothelin-1 or phenylephrine or high potassium buffer. TRPV1 mRNA and protein expression in human arteries were also assessed. TRPV1 transcripts and proteins were detected in human resistance arteries. Capsaicin (1 μM) induced concentration-dependent constriction of endothelium-intact and endothelium-denuded human adipose arterioles (HAA), which was significantly inhibited by SB366791. Pre-constriction of HAA with endothelin-1, but not high potassium buffer or phenylephrine, significantly potentiated capsaicin (0.1 μM)-induced constriction. GF109203X significantly inhibited potentiation of capsaicin-induced constriction by endothelin-1. TRPV1 channels are expressed in the human vasculature and affect vascular tone of human arterioles on activation. Their vasomotor function is modulated by endothelin-1, mediated in part by PKC. These findings reveal a novel interplay between endothelin-1 signalling and TRPV1 channels in human VSMC, adding to our understanding of the ion channel mechanisms that regulate human arteriolar tone and may also contribute to the pathophysiology of coronary vasospasm.

Transglutaminase 2 Inhibitor LDN 27219 Age-Dependently Lowers Blood Pressure and Improves Endothelium-Dependent Vasodilation in Resistance Arteries.

Transglutaminase 2 (TG2) is an enzyme which in the open conformation exerts transamidase activity, leading to protein cross-linking and fibrosis. In the closed conformation, TG2 participates in transmembrane signaling as a G protein. The unspecific transglutaminase inhibitor cystamine causes vasorelaxation in rat resistance arteries. However, the role of TG2 conformation in vascular function is unknown. We investigated the vascular effects of selective TG2 inhibitors by myography in isolated rat mesenteric and human subcutaneous resistance arteries, patch-clamp studies on vascular smooth muscle cells, and blood pressure measurements in rats and mice. LDN 27219 promoted the closed TG2 conformation and inhibited transamidase activity in mesenteric arteries. In contrast to TG2 inhibitors promoting the open conformation (Z-DON, VA5), LDN 27219 concentration-dependently relaxed rat and resistance human arteries by a mechanism dependent on nitric oxide, large-conductance calcium-activated and voltage-gated potassium channels 7, lowering blood pressure. LDN 27219 also potentiated acetylcholine-induced relaxation by opening potassium channels in the smooth muscle; these effects were abolished by membrane-permeable TG2 inhibitors promoting the open conformation. In isolated arteries from 35- to 40-week-old rats, transamidase activity was increased, and LDN 27219 improved acetylcholine-induced relaxation more than in younger rats. Infusion of LDN 27219 decreased blood pressure more effectively in 35- to 40-week than 12- to 14-week-old anesthetized rats. In summary, pharmacological modulation of TG2 to the closed conformation age-dependently lowers blood pressure and, by opening potassium channels, potentiates endothelium-dependent vasorelaxation. Our findings suggest that promoting the closed conformation of TG2 is a potential strategy to treat age-related vascular dysfunction and lowers blood pressure.

AMPK upregulates KCa2.3 channels and ameliorates endothelial dysfunction in diet-induced obese mice

The opening of endothelial small-conductance calcium-activated potassium channels (KCa2.3) is essential for endothelium-dependent hyperpolarization (EDH), which predominantly occurs in small resistance arteries. Adenosine monophosphate-activated protein kinase (AMPK), an important metabolic regulator, has been implicated in regulating endothelial nitric oxide synthase activity. However, it was unclear whether AMPK regulated endothelial KCa2.3-mediated EDH-type vasodilation. Using bioinformatics analysis and myograph system, we investigated the regulation by AMPK of KCa2.3 in human umbilical vein endothelial cells (HUVECs) or mouse second-order mesenteric resistance arteries. In HUVECs, AMPK activation either by activators (AICAR, A769662 and MK-8722) or expression of the constitutively active form of AMPK significantly upregulated KCa2.3 expression. Such effects were abolished by AMPK inhibitor (compound C) or AMPK α1-/α2-siRNA, extracellular-signal-regulated-kinase 5 (ERK5) inhibitor (ERK5-IN-1), and specific siRNA to myocyte-enhancer factor 2 (MEF2) or krüppel-like factor 2/4 (KLF2/4). KCa2.3 expression was significantly reduced in mesenteric resistance arteries in AMPKα2 knockout mice when compared with littermate control mice. Furthermore, in high-fat diet fed mice, 2-week treatment with AICAR restored endothelial KCa2.3 expression in mesenteric resistance arteries with improved endothelial dysfunction. Our results demonstrate that activation of AMPK upregulates KCa2.3 channel expression through the ERK5–MEF2–KLF2/4 signaling pathway in vascular endothelium, which contributes to benefits through KCa2.3-mediated EDH-type vasodilation in mesenteric resistance arteries.