bluuebunny/crossref_metadata_2025 huggingface dataset:
Activators of endothelial calcium‐activated potassium channels enhance the availability of NO released in response to shear stress (1079.22)
Authors: Lunn Stephanie , Govindasamy Natasha , Zwozdesky Myron , Wei Ran , Liew Amanda , Kerr Paul , Plane Frances
Published: June 2021

Nerve‐evoked vasoconstriction of resistance arteries is limited by shear stress‐induced release of endothelium‐derived nitric oxide (NO) and activation of endothelial Ca2+‐activated potassium (KCa) channels. NO and KCa are often regarded as separate pathways for modulation of vascular tone but recent data from our lab indicate small molecule activators of endothelial KCa channels enhance the availability of NO released in response to agonists. In this study we have extended these observations to test the hypothesis that activators of KCa channels can enhance the availability of NO released in response to increases in shear stress caused by nerve‐evoked vasoconstriction.

In the rat perfused mesenteric bed, N‐cyclohexyl‐N‐[2‐(3,5‐dimethyl‐pyrazol‐1‐yl)‐6‐methyl‐4‐pyrimid‐iamine (CyPPA; 1‐10 μM), a small molecule activator of small conductance KCa channels (SKCa) did not alter basal perfusion pressure but did cause endothelium‐ and concentration‐dependent attenuation of nerve‐evoked vasoconstriction. Inhibitors of NO synthase, soluble guanylyl cyclase or SKCa channels, significantly enhanced nerve‐mediated vasoconstriction and prevented the effects CyPPA. In contrast, scavengers of superoxide enhanced the ability of CyPPA to limit vasoconstriction. Inhibition of endothelial intermediate conductance KCa channels or cyclooxygenase did not alter nerve‐evoked responses and did not alter the effects of CyPPA whereas Our findings indicate SKCa channel openers can enhance NO‐mediated modulation of nerve‐evoked vasoconstriction without altering basal perfusion. This finding is significant as in vivo such an effect could reduce vasoconstriction whilst avoiding the baroreceptor activation seen with direct vasodilators and thus may reduce blood pressure whilst maintaining the physiological coupling between nerves, endothelium and changes in blood flow.

Supported by HSFC. SL is supported by a QEII award from the University of Alberta.
Local metabolic and feed forward regulation of coronary blood flow (1079.23)
Authors: Pradhan Ranjan , Beard Daniel , Gorman Mark , Feigl Eric
Published: June 2021

When red blood cells reach a low oxygen environment they release a small amount of ATP into the plasma. The ATP and its breakdown products ADP and AMP act on capillary endothelial purinergic receptors to initiate a vasodilator response that is conducted upstream. This forms the basis of a local metabolic feedback control mechanism where the red blood cell is the sensor of the balance of oxygen delivery (flow) versus oxygen consumption by the heart. This is a paradigm shift from the previous idea that the myocardium acts as the sensor of oxygen balance. The existence of feed forward control of the coronary circulation by sympathetic beta adrenoceptor vasodilation, and a modest alpha adrenoceptor vasoconstriction adds complexity and redundancy. A mathematical computer model has been developed to understand the simultaneous action of feedback and feed forward control of coronary blood flow. Data from canine experiments during control exercise and with adrenergic and purinergic receptor blockade were analyzed to elucidate the simultaneous interplay between local metabolic feedback control, and feed forward coronary control. The results indicate that ~71% of coronary vasodilation is due to purinergic local metabolic control and ~27% is due to feed forward adrenergic control during exercise.

Grant Funding Source: NIH U01 HL 118738
Coronary microvascular dysfunction leads to cardiac dysfunction and development of transient apical ballooning (1079.27)
Authors: Ohanyan Vahagn , Yin Liya , Luli Jordan , Enrick Molly , Stevanov Kelly , Kolz Christopher , Logan Suzanna , Chilian William
Published: June 2021

Transient apical ballooning also known as Takotsubo cardiomyopathy, is a particular acute coronary syndrome and is characterized by transient systolic ballooning of the apex in the absence of significant epicardial coronary disease. The exact mechanism of transient apical ballooning is still unknown, but it is speculated that impaired microvascular metabolic dilation produces the pathology. Previously we found that metabolic dilation (increase in coronary flow in response to augmented cardiac work) was compromised in mice null for Kv1.5 channels (Kv1.5‐/‐) compared to wild type mice (WT). Because compromised flow during metabolic stress (what we observed in the Kv1.5‐/‐ mice) is thought to be an initiating factor for apcial ballooning, we hypothesized that Kv1.5‐/‐ mice, with impaired coronary metabolic dilation, develop apical ballooning when subjected to a chronic metabolic challenge. Transaortic constriction (TAC) was performed to chronically increase afterload in Kv1.5‐/‐ and WT male mice. Measurements of cardiac function were made 7 days after TAC, then at day 10 the constriction was released (de‐banding). Cardiac function was measured up to 6 weeks after de‐banding using high frequency ultrasound. More than 50 % of Kv1.5‐/‐mice developed apical ballooning 7 days after TAC. M‐mode echocardiography was performed at the apex and at the base of the heart to identify ballooning. The apex was significantly dilated compared to the base in Kv1.5‐/‐ mice. During systole the base contracted, but the apex dilated. One week after de‐banding, apical ballooning was absent and 6 weeks later ejection fraction approximated that before TAC. Based on these data, we conclude that impaired coupling of myocardial blood flow to cardiac work, a condition known as coronary microvascular dysfunction, causes apical ballooning during heightened cardiac work.
Dysregulation of pressure‐induced Ca2+ signaling and myogenic constriction of cerebral arteries in aged hypertensive mice (1079.3)
Authors: Toth Peter , Csiszar Anna , Tucsek Zsuzsanna , Sosnowska Danuta , Gautam Tripti , Koller Akos , Schwartzman Michal , Sonntag William , Ungvari Zoltan
Published: June 2021

Hypertension in the elderly substantially increases the risk of stroke and vascular cognitive impairment (VCI) in part due to an impaired functional adaptation of aged cerebral arteries to high blood pressure. To elucidate the mechanisms underlying impaired autoregulatory protection in aging, hypertension was induced in young (3 mo) and aged (24 mo) C57/BL6 mice by chronic infusion of angiotensin II and pressure‐induced changes in smooth muscle (SMC) [Ca2+]i and myogenic constriction of middle cerebral arteries (MCA) were assessed. In MCAs from young hypertensive mice pressure‐induced increases in VSMC [Ca2+]i and myogenic tone were increased and these adaptive responses were inhibited by the cytochrome P450 ω‐hydroxylase inhibitor HET0016 and the TRP channel blocker SKF96365. Administration of 20‐HETE increased SMC [Ca2+]i and constricted MCAs and these responses were inhibited by SKF96365. MCAs from aged hypertensive mice did not show adaptive increases in pressure‐induced calcium signal and myogenic tone and responses to HET0016 and SKF96365 were blunted. Inhibition of BK channels by iberiotoxin enhanced SMC [Ca2+]i and myogenic constriction in MCAs of young normotensive animals, whereas it was without effect in MCAs of young hypertensive mice. Iberiotoxin did not restore myogenic adaptation in MCAs of aged hypertensive mice. Thus, functional maladaptation of aged cerebral arteries to hypertension is due to the dysregulation of pressure‐induced, 20‐HETE and TRP channel‐mediated SMC calcium signaling, whereas overactivation of BK channels is unlikely to play a role in this phenomenon.

Grant Funding Source: Supported by the American Heart Association and the Nemzeti Fejlesztési Ügynökség
Aging exacerbates hypertension‐induced intracerebral microbleeds in mice (1079.6)
Authors: Tarantini Stefano , Toth Peter , Sosnowska Danuta , Gautam Tripti , Tucsek Zsuzsanna , Koller Akos , Sonntag William , Csiszar Anna , Ungvari Zoltan
Published: June 2021

Aging is associated with high prevalence of cerebral microbleeds (CMBs) in the elderly, which contribute to the age‐related decline in higher cortical function. Despite its clinical significance, the pathophysiology of CMBs is poorly understood. To establish a novel model of CMB hypertension was induced in young (3 mo) and aged (24 mo) C57B/6 mice (s.c AngII, p.o. L‐NAME). Neurological examination and gait analysis followed by histological analysis of CMBs in serial sections of the brain showed that in the aged mice the same level of hypertension lead to significantly earlier onset and increased incidence of CMBs (incidence: young:27%, aged:90% of the animals; average number of CMBs: young:15±3, aged:28±2). Aging exacerbated hypertension‐induced cerebral oxidative stress, NADPH oxidase expression (Nox2 mRNA: 2±0.1 fold increase in aged vs. young) and activation of MMPs (6±0.9 fold increase in aged vs. young). Treatment of aged mice with the dietary polyphenol resveratrol (200 mg/kg for 20 days) significantly attenuated oxidative stress, down‐regulated NADPH oxidase, decreased MMP activity and prevented/delayed the development of CMBs. Collectively, aging exacerbates hypertension‐induced intracerebral microbleeds in mice likely by increasing oxidative stress and MMP activation. Therapeutic strategies to reduce microvascular oxidative stress and MMP activity should be considered for the prevention of CMBs in the elderly.

Grant Funding Source: Supported by the Oklahoma Center for the Advancement of Science and Technology
The R403Q mutation alters isometric and energetic properties in 2 month mice (1081.1)
Authors: Birch Camille , Konhilas John
Published: June 2021

Familial hypertrophic cardiomyopathy is a primary disease of the sarcomere. The R403Q mutation resides at the actin‐interaction site on myosin and leads to progressive hypertrophic cardiomyopathy that can lead to sudden cardiac death or congestive heart failure. We hypothesized that the R403Q mutation intrinsically increases the energetic cost of contraction.

To do this, we assessed Ca2+‐sensitivity of tension and cross‐bridge kinetics in de‐membranated cardiac trabeculae from wild‐type (WT) and R403Q hearts at 2 months of age. We measured cross‐bridge kinetics using 2 methods: (1) the rate of ATP hydrolysis (off‐rate) by NADH oxidation, and (2) the rate of force redevelopment (ktr) by a rapid release‐restretch protocol.

Male R403Q mice display a decrease in Ca2+ sensitivity compared to WT males and females at SL 2.0μm. R403Q males display increased tension cost at SL 2.0μm when compared to WT counterparts. At maximal activation, the rate of force redevelopment is not significantly different between R403Q and WT males. Myosin heavy chain isoform gel analysis reveals no difference in β‐myosin content between R403Q mice and WT controls.

Although maximal ktr is not significantly different between WT and R403Q males, an increase in ATP hydrolysis suggests an alteration in binding kinetics. Along with no overt pathology at 2 months, the R403Q mutation alters the energetic cost of contraction and isometric properties.

Grant Funding Source: Supported by R01 HL098256A and T32HL007955
The influence of estrogen on circadian rhythms of heart rates and cardiac sympathetic nerve in female rats (1083.6)
Authors: Marui Shuri , Matsuda Mayumi , Sato Nobuo , Nagashima Kei
Published: June 2021

Purpose We aimed to clarify the effect of estradiol (E2) on heart rates (HR)and arterial pressure (AP), and mechanisms. Methods Female Wistar rats (n=9, age of 7‐9 wk) were bilaterally ovariectomized, and placed a radio transmitter for HR and AP measurements in the abdominal cavity. Two silicon tubes containing E2 were s.c. placed in one group (n=4, E2(+)), and empty tubes for the other (n=5, E2(‐)). The tubes were removed 10 days after the surgery (Day 0). On Day 7 or 21, rats were killed. The left ventricle of heart was taken, and protein contents of β1 and β2‐adrenoreceptors (AR) was determined by Western blotting. Results On Day 0, HR was greater (P<0.05) in the E2(‐) than that in the E2(+) group (388±15 and 337±13 beats/min (bpm) in the light phase; and 450±12 and 390±12 bpm in the dark phase, respectively). On Day 14, HR in the E2(+) group increased (377±15 bpm in the light phase; and 431±14 bpm in the dark phase). On Day 21, HR became lower (P<0.05) in the E2(‐) group (330±17 bpm in the light phase; and 388±20 bpm in the dark phase). Mean AP was not different between the two groups on each day. Both β1‐AR and plasma noradrenaline were greater (P<0.05) in the E2(‐) than E2(+) group, on Day 0. On Day 7, both β1‐AR and plasma noradrenaline decreased, however on Day 21, plasma noradrenaline became higher again. Conclusion A reduction of plasma estrogen level may affect HR, which is resulted from greater sympathetic activity and β1‐AR expression in the heart.

Grant Funding Source: This study was supported by MEXT. KIBANKEISEI
Co‐releasing molecules attenuate placental ischemia‐induced hypertension in pregnant rats (1084.4)
Authors: George Eric , Stec David , Granger Joey
Published: June 2021

Preeclampsia is a serious complication of pregnancy marked by hypertension and maternal endothelial dysfunction. Recently we have published a series of studies demonstrating that induction of heme oxygenase‐1 (HO‐1) could attenuate many of the maternal symptoms of preeclampsia in a rodent model of placental ischemia induced by Reductions on Uterine Perfusion Pressure (RUPP). We have further shown that the individual metabolites of HO‐1, namely CO and bilirubin, both downregulate hypoxia‐induced production of known placental derived pathogenic factors ex vivo. Here, we tested whether , administration of the CO‐releasing molecule CORM‐3 could attenuate the maternal symptoms of placental ischemia. In response to placental ischemia, rats exhibited marked increases in blood pressure compared to control animals (107 ± 1 vs 136 ± 2mmHg, p<0.05). Interestingly, administration of the CO‐releasing molecule CORM‐3 (5mg/kg i.v., qd) significantly decreased blood pressure in both normal pregnant (95 ± 2mmHg) and RUPP (113 ± 1) animals compared to their respective controls. Interestingly, while CORM‐3 had no significant effect on placental VEGF levels, the placental level of the VEGF antagonist sFlt‐1 was markedly increased in control (596 ± 45 vs 1143 ±122 pg/mg, p<0.05) animals, and trended towards increases in RUPP animals (1004 ± 81 vs 1213 ± 72 pg/mg) versus respective controls. This was accompanied by a significant decrease in circulating free VEGF in both Control (527 ± 30 vs 294 ± 9 pg/ml, p<0.05) and RUPP groups (363 ±44 vs 255 ± 23 pg/ml) suggesting that restoration of circulating angiogenic balance in these animals is not the mechanism responsible for their decreased blood pressure.

Grant Funding Source: Supported by HL‐108618, HL‐51971 (JPG), HL‐088421, HL‐088421‐S1 (DES), and HL116774 (EMG).
The role of TNF alpha in placental ischemia‐induced cerebrovascular abnormalities (1084.6)
Authors: Warrington Junie , Drummond Heather , Ryan Michael , Granger Joey
Published: June 2021

While cerebrovascular events contribute to 40% of preeclampsia/eclampsia‐related deaths, the underlying contributing factors are unclear. Circulating tumor necrosis factor α (TNFα) is elevated in women with preeclampsia. We tested the hypothesis that TNFα contributes to cerebrovascular abnormalities that can be prevented by TNFα blockade. TNFα infusion in pregnant rats (100 ng/day ip, from gestational day 14 to 19) increased mean arterial pressure (MAP; 109 ± 3 vs. 99 ± 1 mmHg, p<0.05). Brain water content increased from 78.6 ± 0.1 to 79.0 ± 0.1% in TNFα‐infused rats (p<0.05), mainly due to increases in the anterior brain (79.6 ± 0.3 vs. 78.9 ± 0.1%; p<0.05). TNFα infusion increased hippocampal blood‐brain barrier (BBB) permeability, assayed using Evans blue extravasation, by 89.8 ± 27.2% (p<0.05). To assess the role of endogenous TNFα, a rat placental ischemia model of preeclampsia, induced by reducing uterine perfusion pressure, was treated with the soluble TNFα receptor, etanercept (0.8 mg/kg, sc.) on gestational day 18. Etanercept reduced placental ischemia‐mediated increases in 1) MAP (121 ± 3 to 115 ± 2 mmHg, vs.102 ± 2 in normal pregnant group; p<0.05), 2) anterior brain water content (80.1 ± 0.4 to 79.0 ± 0.2%, vs. 79.2 ± 0.2% in normal pregnant group; p<0.05), and 3) BBB permeability (202 ± 44 to 101 ± 28% of normal pregnant rats). Our results indicate that TNFα may mediate cerebral edema by increasing BBB permeability in response to placental ischemia. Thus, TNFα may be a target for preventing cerebrovascular complications associated with preeclampsia.

Grant Funding Source: Supported by: NIH R01 HL108618, PPG HL51971 and AHA 13POST16240000
Complement C5a receptor antagonist attenuates placental ischemia‐induced hypertension in rat (1084.7)
Authors: Peterson Jenna , Lillegard Kathryn , Elmquist Barbara , Gilbert Jeffrey , Regal Jean
Published: June 2021

Preeclampsia is a pregnancy specific condition characterized by decreased placental perfusion, new onset hypertension, intrauterine growth restriction, decreased free plasma vascular endothelial growth factor (VEGF) and increased innate immune complement system activation producing C3a and C5a. Our previous work demonstrated the importance of complement activation in the reduced uterine perfusion pressure (RUPP) model of placental ischemia‐ induced hypertension in rat. However, the complement activation product important in mediating the hypertension is unclear. Hypothesis: Complement activation product C5a mediates placental ischemia‐induced hypertension independent of changes in free plasma VEGF. On gestation day (GD) 14, rats underwent sham surgery or clip placement on ovarian arteries and inferior abdominal aorta (RUPP) resulting in placental ischemia and increased blood pressure on GD19. The role of C5a was determined by once daily injection of C5a receptor antagonist acetyl‐F‐[Orn‐P‐(D‐Cha)‐WR] (PMX53, 3mg/kg subcutaneous) or vehicle (10% ethanol in saline) on GD14‐18. On GD19, mean arterial pressure (MAP, mmHg) was measured via arterial catheter. Complement activation was monitored by measuring relative serum C3a concentration since C5a was not detectable in serum by current assay methods. VEGF was assessed by ELISA. MAP increased in RUPP (n=23, 108±2) vs. Sham (n=19, 93±2) and PMX53 treatment significantly decreased MAP in RUPP rats (n=11, 101±3) with no effect in Sham (n=5, 93±3). As expected, complement activation increased in RUPP vs Sham (p<0.05). Also, fetal weight and VEGF decreased in RUPP vs Sham (p<0.05). PMX53 treatment did not reverse RUPP‐induced changes in complement activation, fetal weight, or VEGF. These data indicate that C5a in part mediates placental ischemia‐induced hypertension independent of reduced free plasma VEGF.

Grant Funding Source: Supported by NIH HL109843
A new model of intrauterine growth restriction (IUGR) induced by reduced uterine perfusion in the mouse programs hypertension in the IUGR mouse offspring (1085.1)
Authors: Intapad Suttira
Published: June 2021

Blood pressure is inversely related to birth weight suggesting that cardiovascular disease can be programmed by exposure to an adverse environment during early life. Numerous experimental models of developmental insults are established in the rat; yet, few mouse models are used. Thus, the aim of this study was to develop a model of intrauterine growth restriction (IUGR) in the mouse for study of mechanisms linking birth weight and hypertension. Pregnant C57BL/6J mice were subjected to either a sham surgery (control offspring) or a mechanical reduction of blood flow to the abdominal aorta and both branches of ovarian arteries initiated at 13 day of gestation to induce IUGR. Birth weight of the IUGR offspring was significantly lower than control offspring (1.23±0.03 vs. 1.42±0.03 g, P<0.002; IUGR vs. control, respectively). Litter size did not differ between groups. Mean arterial blood pressure measured in conscious mice chronically instrumented with a carotid arterial catheter was significantly elevated in IUGR versus age‐matched control offspring at 5 weeks of age (110±3 vs. 92±17 mmHg, P<0.001; respectively). Thus, these data suggest that reduced uterine perfusion in the mouse programs IUGR‐induced hypertension. Importantly, the use of a mouse model of IUGR will allow for site and conditional genetic modifications to address novel hypotheses related to the developmental origins of hypertension.
Blockade of the renin angiotensin system abolishes age‐dependent hypertension in female intrauterine growth restricted rats in the absence of an enhanced sensitivity to acute angiotensin II (1085.2)
Authors: Intapad Suttira , Dasinger John , Backstrom Miles , Alexander Barbara
Published: June 2021

Low birth weight (LBW) as an indicator of poor fetal growth is a risk factor for hypertension. Placental insufficiency induces intrauterine growth restriction (IUGR) in the female rat indicative of LBW. Female IUGR rats exhibit an enhanced sensitivity to acute angiotensin II (Ang II) while remaining normotensive under basal conditions at 4 months of age. Yet, female IUGR rats develop hypertension by 12 month of age. Thus, this study tested the hypothesis that the renin angiotensin system (RAS) contributes to the etiology of age‐dependent hypertension in the female IUGR rat. At 12 months of age rats were treated with vehicle or the ACE inhibitor, enalapril (250 mg/L in drinking water ad libitum), for 2 weeks then mean arterial pressure (MAP) was measured in conscious, chronically instrumented rats. MAP was significantly higher in untreated IUGR versus untreated control rats (134±3 vs. 117±4 mmHg, P<0.05; respectively); enalapril abolished the difference in MAP (118±2 vs. 113±1 mmHg; respectively). Acute infusion (20 min) of Ang II (100 ng/kg/min) increased MAP in all groups (155±3, 164±2, 148±1, 154±2 mmHg; control untreated, IUGR untreated, control treated & IUGR treated; respectively). Yet, the increase in MAP to Ang II was not enhanced in IUGR versus control counterpart. Thus, these data suggest that the RAS contributes to age‐dependent hypertension in the female IUGR in the absence of enhanced sensitivity to Ang II.

Grant Funding Source: Supported by HL074927, HL51971, 12POST11980021
Early life stress induces circulating factor(s) promoting endothelial dysfunction (1085.3)
Authors: Ho Dao , Burch Mariah , Musall Benjamin , Pollock Jennifer
Published: June 2021

In humans, early life stress (ELS) increases cardiovascular disease (CVD) risk and elevates circulating pro‐oxidant factors; however the relationship between ELS‐induced CVD and circulating pro‐oxidant factors is not known. In a mouse model of ELS, maternal separation with early weaning (MSEW), we previously showed that MSEW induces adult endothelial dysfunction. We hypothesized that MSEW promotes endothelial dysfunction by increasing circulating factors that increase superoxide (O2‐) production and reduce NO bioavailability in endothelial cells. MSEW litters underwent maternal separation during postnatal days (PD) 2‐16 for 4‐8h/day, and weaning at PD17. Control (CON) litters remained undisturbed and were weaned at PD21. To determine the impact of MSEW‐induced circulating factors on endothelial function, we incubated Mouse Aortic Endothelial Cells (MAECs) with plasma of CON or MSEW mice (25 ul plasma/ml media; 24 hr). MSEW plasma‐treated MAECs had increased O2‐ levels compared to CON (dihydroethidium‐HPLC; CON = 25.7 + 9.7 vs MSEW = 107.0 + 18.5 pmol/mg pr/hr; p < 0.001). Apocynin (300 μM; NADPH oxidase inhibitor) abolished this effect. However, no change in NO bioavailability (nitrite determination by HPLC) was found. In conclusion, MSEW may promote cardiovascular disease risk by inducing circulating factors that enhance endothelial O2‐ production in an NADPH oxidase‐dependent manner.

Grant Funding Source: Supported by NIH grants 1F32 HL116145; P01 HL69999
Maternal western diet primes non‐alcoholic fatty liver disease in adult offspring in mice (1085.4)
Authors: Plosch Torsten , Pruis Maurien , Lendvai Agnes , Zwier Mathijs , de Bruin Alain , Groen Albert K.
Published: June 2021

Metabolic programming via the maternal diet during gestation may play a role in the development of different aspects of the metabolic syndrome. Using a mouse model we aimed to characterise the role of maternal western type diet in the development of non‐alcoholic fatty liver disease (NAFLD) in the offspring. Female mice were fed either a western (W) or low‐fat control (L) semi‐synthetic diet before and during gestation and lactation. At weaning, male offspring were assigned either the W or the L diet, generating four experimental groups: WW, WL, LW and LL offspring. Biochemical, histological and epigenetic indicators were investigated at 29 weeks of age. Male offspring exposed to prenatal and post‐weaning western‐style diet (WW) showed hepatomegaly combined with accumulation of hepatic cholesterol and triglycerides. This was associated with up‐regulation of de novo lipid synthesis, inflammation and dysregulation of lipid storage. Histopathological analysis revealed the presence of advanced steatohepatitis in WW offspring. In addition, alterations in DNA methylation in key metabolic genes (Ppara, Insig, and Fasn) were detected. In conclusion, maternal dietary fat intake during early development programs susceptibility to liver disease in male offspring, mediated by disturbances in lipid metabolism and inflammatory response. Long lasting epigenetic changes may underlie this dysregulation.

Grant Funding Source: Supported by CTMM and NHS
Reduced uterine perfusion pressure alters angiogenic balance in the amniotic fluid and fetal lung of the rat (1085.9)
Authors: Rear Alice , Needham Karen , Lovering Andrew , Regal Jean , Gilbert Jeffrey
Published: June 2021

Placental ischemia, angiogenic imbalance in the maternal circulation, preterm birth and very low birth weight are characteristic features of preeclampsia. Although recent studies suggest preeclampsia increases risk of the hypoplastic lung disease bronchopulmonary dysplasia (BPD), whether or not chronic placental ischemia results in fetal angiogenic imbalance remains unclear. We hypothesized chronic RUPP would result in decreased expression of angiogenic proteins in the amniotic fluid and fetal lung consistent with the development of BPD. RUPP was induced on gestation day (GD) 14 and fetal lung tissue and amniotic fluid were collected on GD 19 from RUPP and normal pregnant (NP) rats. Fetuses were smaller (1.8 ± 0.19 vs. 2.3 ± 0.08 g; P<0.05) and amniotic fluid free vascular endothelial growth factor (VEGF; 12.6±0.82 vs. 9.19±1.29 pg/ug; P<0.05) decreased in RUPP compared to NP. Soluble fms‐like tyrosine kinase (sFlt‐1) to VEGF ratio was elevated (12.6±0.82 vs. 9.19±1.29; P<0.05) in RUPP versus NP amniotic fluid. VEGF receptor‐2 was decreased (48%; P< 0.05) in lung tissue from RUPP compared to NP fetuses. These data show that chronic placental ischemia results in angiogenic imbalance in fetal tissues similar to that previously observed in the maternal circulation by our group and others. Furthermore, these findings are consistent with the hypothesis that angiogenic imbalance in utero plays an important role in the abnormal vasculogenesis and alveologenesis associated with very low birth weight infants.

Grant Funding Source: AHA, NIH
Coordinated regulation of ENaC activity in the distal nephron by aldosterone and Ang II (1088.14)
Authors: Mamenko Mykola , Zaika Oleg , Prieto Minolfa , Jensen Behrana , Doris Peter , Navar Gabriel , Pochynyuk Oleh
Published: June 2021

Recent studies point to direct actions of Ang II on ENaC‐mediated Na+ reabsorption in the distal nephron via stimulation of AT1R that occurs independently of aldosterone‐MR signaling. We used split‐opened distal nephrons from mice to probe the interaction between aldosterone and Ang II cascades in controlling ENaC activity during variations in dietary salt intake and in Ang II‐induced hypertension. Systemic MR inhibition abolished increases in the numbers of functionally active ENaC in response to low Na+ diet. Simultaneous inhibition of MR and AT1R at low dietary salt intake not only reduced the amount of active channels, but also decreased ENaC open probability and produced significantly greater natriuresis than either treatment alone. Chronic Ang II infusion stimulated ENaC activity greatly above levels observed during dietary sodium restriction. Importantly, maximal MR inhibition failed to suppress ENaC activity to control values. In contrast, blocking of de novo Ang II synthesis with captopril abolished stimulation of ENaC activity in Ang II infused animals. We conclude that during variations in dietary salt intake both aldosterone and Ang II contribute complementarily to the regulation of ENaC activity. In contrast, aldosterone‐independent stimulation of ENaC activity mediated by ACE dependent endogenous intratubular Ang II formation predominates in the setting of Ang II‐induced hypertension.

Grant Funding Source: DK095029
Relationship between renal Ang (1‐7) induced sodium and water excretion and AT2 receptors (1088.7)
Authors: O’Neill Julie , Johns Edward
Published: June 2021

Ang (1‐7) causes a natriuresis, which is mediated by intrarenal (IR) Mas receptors but is also dependent upon functional IR AT1 receptors. This study investigated the role played by IR AT2 receptors in mediating the renal excretory actions of Ang (1‐7) normally and when the RAS was stimulated by a low sodium diet. Male Wistar rats (n= 5‐6) were placed on normal (NNa+, 0.2%) or low (LNa+, 0.03%) sodium diets for 2 weeks. Rats were anaesthetized (chloralose/urethane) and prepared for mean arterial pressure (MAP) and left renal function measurement and IR infusion of saline and drugs. Data ±SEM were subjected to a Students t‐test. Basal MAP was 93±4 and 91±2mmHg and basal fractional sodium excretion (FENa) was 1.4±0.3 and 0.7±0.2 % in NNa+ and in LNa+ rats, respectively. IR infusion of Ang1‐7 had no effect on MAP but increased FENa by 227% and 427% in NNa+ and LNa+ rats respectively (both P<0.05). In the presence of PD123319, Ang (1‐7) induced increases in FENa excretion were blunted at 75% and 34% in NNa+ and LNa+ rats respectively (both P<0.05). These data imply that functional IR AT2 receptors may have a permissive effect upon the natriuretic actions of Ang (1‐7) at a tubular level.

Grant Funding Source: IRCSET
Newly identified caveolin‐1 mutation associated with heritable human pulmonary arterial hypertension mediates hyperproliferation via augmented calcium signaling (1089.10)
Authors: Marsboom Glenn , Theriault Kraig , Debroy Auditi , Mao Mao , Baig Saqib , Tiruppathi Chinnaswamy , Bonini Marcelo , Rehman Jalees , Austin Eric , Malik Asrar
Published: June 2021

Objective: Recently, a new mutation in Caveolin‐1 (Cav‐1) was identified in a family with heritable pulmonary arterial hypertension (hPAH), but the underlying molecular mechanisms by which the Cav‐1 mutation leads to hPAH are poorly understood.

Methods: Human dermal fibroblasts were obtained from 3 healthy control subjects and 3 hPAH patients with the Caveolin‐1 mutation.

Results: Patient fibroblasts had a markedly higher proliferative rate than those of control subjects (34±5 vs. 19±4%, P<0.05). Since calcium is an important regulator of cell proliferation, we compared maximal cytoplasmic calcium influx in response to the SERCA inhibitor thapsigargin. We found that calcium influx was higher in fibroblasts with a Cav‐1 mutation (2.38‐fold increase, P<0.005). Also, fibroblasts with a Cav‐1 mutation exhibited higher oxidative stress (2.5‐fold increase in H2O2 P<0.01, 4‐fold increase in peroxynitrite P<0.001).

Conclusions: The newly identified Caveolin‐1 in hPAH patients as associated higher fibroblast proliferation, aberrant calcium signaling, and high levels of oxidative stress. These signaling pathways may be promising targets for novel therapies in hPAH and validate the role of Cav‐1 in clinical PAH. Since Cav‐1 is a disease modifier in experimental PAH, our findings may also prove valuable for understanding the pathophysiology of non‐heritable PAH.
Enhanced endothelin‐1‐ and depolarization‐induced pulmonary vasoconstriction following chronic hypoxia require Src family kinases (1089.12)
Authors: Norton Charles , Walker Benjimen , Resta Thomas
Published: June 2021

Chronic hypoxia (CH) augments depolarization‐induced pulmonary vasoconstriction through an epidermal growth factor receptor (EGFR)/NADPH oxidase (NOX)/Rho kinase (ROK)‐dependent Ca2+ sensitization pathway. Considering the importance of Src kinases as effectors of both endothelin receptor (ETR) and EGFR signaling, we hypothesized that Src kinases provide a common signaling moiety that couples depolarization and ETR stimulation to enhanced EGFR\NOX‐mediated pulmonary vasoconstriction following CH. To test this hypothesis, we measured constriction to increasing concentrations of KCl and endothelin‐1 (ET‐1) in endothelium‐disrupted, Ca2+ permeabilized pulmonary arteries (~150 μm diameter) from control and CH (4 wk at 0.5 atm) rats. Inhibition of EGFR (AG 1478, 1 µM), NOX (gp91ds‐tat, 50 µM), or the NOX component Rac1 (NSC 23766, 50 µM) attenuated constriction to ET‐1 in arteries from CH but not control rats (p<0.05). Consistent with our hypothesis, enhanced KCl‐ and ET‐1‐induced constriction following CH were prevented by Src inhibition (Su 6656, 10 µM). In contrast, direct stimulation of EGFR with epidermal growth factor caused constriction only in arteries from CH rats, which was blocked by inhibition of NOX or ROK (HA‐1077, 10 µM) but not Src. We conclude that Src couples depolarization and ETR stimulation to EGFR/NOX‐mediated pulmonary vasoconstriction following CH.
Rosiglitazone attenuated endothelin‐1‐induced vasoconstriction of pulmonary arteries in the rat model of pulmonary arterial hypertension (1089.13)
Authors: Liu Yahan , Tian XiaoYu , Huang Yu , Wang Nanping
Published: June 2021

Pulmonary arterial hypertension (PAH) can lead to right ventricular failure and death. Endothelin (ET)‐1 activation has been demonstrated in plasma and lung tissue of PAH patients and animal models. Peroxisome proliferator‐activated receptor γ (PPARγ) agonists have been shown to ameliorate PAH. To investigate the mechanism for anti‐vasoconstrictive effects of rosiglitazone in PAH, Sprague‐Dawley rats were exposed to chronic hypoxia (10% oxygen) for 3 weeks. ET‐1 caused enhanced constriction of pulmonary arteries from such PAH rats. Oral gavage with PPARγ agonist rosiglitazone (20 mg/kg per day) for 3 days in vivo reversed the vasocontractive effect of ET‐1. This effect was abolished by L‐NAME, indicating a NO‐dependent mechanism. The attenuated ET‐1‐induced contraction of rosiglitazone was restored by an ETBR antagonist A192621, whereas this antagonist did not modulate the evoked pulmonary arterial contractions from normoxia and PAH rats. Rosiglitazone increased protein level of ETBR in pulmonary arteries from PAH rats. These results demonstrated that rosiglitazone attenuated ET‐1‐induced vasoconstriction in PAH through the upregulation of endothelial ETBR and suggested the therapeutic use of PPARγ agonists in PAH.

Grant Funding Source: Supported by Grants from the National Science Foundation of China (81220108005, 81300189)
Actin polymerization contributes to enhanced pulmonary arterial vasoconstrictor reactivity following chronic hypoxia (1089.15)
Authors: Sheak Joshua , Sands Michelle , Snow Jessica , Gonzalez Bosc Laura , Jernigan Nikki , Walker Benjimen , Resta Thomas
Published: June 2021

Chronic hypoxia (CH) induces basal pulmonary arterial tone and augments vasoconstrictor reactivity to endothelin‐1 (ET‐1) through Rho kinase‐dependent Ca2+ sensitization. Considering that dynamic actin cytoskeletal reorganization is integral to tension generation in smooth muscle, and that Rho kinase is important in mediating actin polymerization, we hypothesized that actin polymerization is required for both basal pulmonary arterial tone and enhanced ET‐1‐dependent vasoconstriction following CH. To test this hypothesis, we assessed effects of the actin polymerization inhibitors, cytochalasin B (10‐5 M) and latrunculin A (10‐5 M), on the development of tone in response to increasing intraluminal pressure steps (5‐45 mmHg) in isolated, endothelium‐disrupted pulmonary arteries (~150 μm diameter) from control and CH (4 wk at 0.5 atm) rats. Further experiments examined effects of cytochalasin B on constrictor responses to ET‐1 (10‐10 ‐ 10‐7 M) under Ca2+‐clamp conditions in arteries from each group. Consistent with our hypothesis, inhibitors of actin polymerization prevented basal tone (p<0.05 vs. vehicle) and attenuated ET‐1‐mediated constriction (p<0.05 vs. vehicle) in arteries from CH rats, while having no effect on control arteries. We conclude that actin polymerization is necessary for generation of basal pulmonary arterial tone and enhanced vasoconstrictor reactivity to ET‐1 following CH.

Grant Funding Source: Supported by NIH R01 HL88192 and NIH T32 HL07736
The role of mitochondrial reactive oxygen species in hypoxia‐induced pulmonary hypertension (1089.16)
Authors: Adesina Sherry , Hart C. , Sutliff Roy
Published: June 2021

Oxidative stress plays a critical role in pulmonary hypertension (PH) pathogenesis. The specific reactive oxygen species (ROS) responsible have not been defined, though mitochondrial ROS likely contribute. We hypothesize that mitochondrial O2•− and H2O2 promote PH pathogenesis.

Hypoxia effects on mitochondrial O2•− and H2O2 were measured using mitoPY1 and mitoSOX in human pulmonary arterial endothelial cells (HPAECs) exposed to normoxia (21% O2) or hypoxia (1% O2) for 72 hours. Mouse models with mitochondria‐targeted SOD2 (TghSOD2) or catalase (mCAT) expression were used to modulate mitochondrial ROS. Control and transgenic mice were exposed to normoxia (21% O2) or hypoxia (10% O2) for 3 weeks. ROS levels (confocal microscopy and Amplex red assay), Nox mRNA levels (qRT‐PCR), and proliferation and remodeling (α‐smooth muscle actin, α‐SMA, staining) were measured at the conclusion of the study.

Hypoxia exposed HPAECs had increased mitochondria‐derived O2•− and H2O2 (p 蠄 0.5). Compared to control, mCAT expression prevented hypoxia‐induced increases in lung H2O2, Nox2, Nox4, and PCNA mRNA, and α‐SMA staining (p 蠄 0.5). Hypoxia‐induced alterations in these markers were exacerbated in TghSOD2 lungs (p 蠄 0.5). These studies suggest that targeted attenuation of mitochondria‐derived H2O2 attenuates pulmonary vascular derangements involved in PH pathogenesis.

Grant Funding Source: VA Merit Review 1I01BX001910. NIH grants DK074519/ HL102167 (CMH/RLS). NRSA 1F31HL114386 ‐ 01A1 SEA
Implication of miR‐204/RUNX2‐dependent calcification pathway in pulmonary arterial hypertension (1089.23)
Authors: Pflieger Aude , Meloche Jolyane , Tremblay Eve , Breuils‐Bonnet Sandra , Provencher Steeve , Bonnet Sebastien
Published: June 2021

Introduction: Pulmonary arterial hypertension (PAH) is a vascular remodeling disease characterized by occurrence of several vascular lesions. Interestingly, some biological pathways implicated in these lesions are also implicated in calcification processes. Nonetheless, the role for calcification lesions in PAH has never been explored. miR‐204, which is downregulated in PAH, is implicated in calcification processes by directly regulating the pro‐calcification transcription factor RUNX2. RUNX2 also regulates cell proliferation by activating HIF‐1. Thus, we hypothesized that miR‐204 downregulation in PAH triggers RUNX2 expression promoting calcification and vascular lesions in PAH. Methods and results: Using human explanted lungs, fresh distal pulmonary arteries (PAs) and freshly isolated pulmonary artery smooth muscle cells (PASMC) from both control and PAH patients, we demonstrated a significant downregulation of miR‐204 and an upregulation of RUNX2 in PAH compared to controls. The increase in RUNX2 in PAH patients was associated with an increase in distal PA calcification. Gain‐ and loss‐of‐function approaches in human PASMC showed that downregulation of miR‐204 in control PASMC increases RUNX2 expression, while increased miR‐204 in PAH‐PASMC decrease it. More importantly, restoring miR‐204 or decreasing RUNX2 expression in PAH‐PASMC decreases proliferation, resistance to apoptosis and alkaline phosphatase activity. Finally, in Sugen/hypoxia PAH rat model, nebulization of either miR‐204 mimic or RUNX2 siRNA significantly improves PAH.

Conclusion: Taken together, our study uncovers a new miR‐204/RUNX2 axis contributing to both calcified lesions and vascular remodeling in distal PA of PAH patients. More experiments are required to further explore the role of RUNX2 in PAH pulmonary artery calcification. This study might lead to the discovery of new potential therapeutic strategies for patients suffering of PAH.
Augmented pulmonary artery pressure response during exercise in adults born extremely preterm, but not in those with bronchopulmonary dysplasia (1089.4)
Authors: Laurie Steven , Elliott Jonathan , Beasley Kara , Mangum Tyler , Goodman Randall , Gladstone Igor , Lovering Andrew
Published: June 2021

Advances in perinatal care in the post surfactant era have increased survivability of infants born extremely preterm (<32 weeks gestation), yet long‐term cardiopulmonary consequences of prematurity in adulthood remain largely unexplored. We studied adults who developed new bronchopulmonary dysplasia (BPD, 22.7 yrs), preterm subjects who did not develop BPD (PRE, 21.1 yrs), and fullterm controls (CONT, 22.7 yrs) at rest and during cycle ergometer exercise in the forward leaning aerobar position. Subjects exercised for 3 min at 25%, 50% and 75% of their maximum workload attained at VO2max from a separate visit. We measured metabolic rate, and pulmonary artery systolic pressure (PASP) with ultrasonography, at rest and during exercise. There were no differences in PASP between the groups at rest. At 50% and 75% VO2max, PASP was higher in PRE subjects than in BPD and CONT subjects, which were not significantly different from each other at any exercise intensity. The slope of the PASP to VO2 relationship was significantly greater in PRE subjects compared to BPD and CONT. These data suggest PRE subjects have a reduced ability for the pulmonary vasculature to accommodate increases in cardiac output during exercise compared to CONT and BPD subjects. Further research is needed to determine if this augmented pressure response during exercise may predispose PRE subjects to developing pulmonary arterial hypertension.

Grant Funding Source: Supported by: AHA Scientist Development Grant
TRPV4‐associated alterations of Ca2+ pulsars in a murine model of pulmonary hypertension secondary to heart failure (1090.11)
Authors: Dayeh Nour , Safar Steven , Gillis Marc‐Antoine , Blanchette Alexandre , Dupuis Jocelyn , Ledoux Jonathan
Published: June 2021

Heart failure is the most frequent cause of pulmonary hypertension (PH) (group 2). Pathophysiological mechanisms modulating pulmonary vascular tone and leading to group 2‐PH are poorly defined. Since Ca2+ homeostasis is crucial to endothelial control of vascular tone, mechanisms involved, including TRPV4 channels, may be altered. This study aimed to characterize intracellular Ca2+ dynamics in pulmonary endothelium and their alterations in group 2 PH. A mouse model of group 2 PH was developed and 3rd order resistance arteries were isolated to investigate endothelial intracellular Ca2+. TRPV4 involvement was studied using a high‐speed confocal microscope. Characterization of endothelial localized calcium transients, similar to Ca2+ pulsars, was carried out in pulmonary arteries from control mice. Exposure of Sham arteries to GSK1016790A (100 nM), a TRPV4‐agonist, increased the frequency (1.5 fold) of Ca2+ pulsars. However, exposure to GSK1016790A had little if any effect on Ca2+ dynamics in PH arteries. TRPV4 channel expression at both mRNA and protein levels within pulmonary endothelium was confirmed by qPCR, IHC and WB approaches. Our data suggest a potential role for endothelial TRPV4 in vasoregulatory alterations involving local Ca2+ dynamics. This study strengthens our understanding of endothelial Ca2+ dyshomeostasis occurring in a clinically relevant model of PH.

Grant Funding Source: FRQS, HSFC, FICM, CIHR
Shear stress enhance intracellular Ca2+ in PASMC from patients with idiopathic pulmonary arterial hypertension (1090.13)
Authors: Song shanshan , Tang Haiyang , Smith Kimberly , Makino Ayako , Yuan Jason
Published: June 2021

Idiopathic pulmonary arterial hypertension (IPAH) is a fatal and progressive disease which is attributed to elevated pulmonary arterial pressure and pulmonary vascular resistance due in part to sustained pulmonary vasoconstriction and vascular remodeling. Pulmonary arterial smooth muscle cells (PASMC) are normally shielded from exposure to shear stress and sense small flow shear through transmural interstitial flow driven by transvascular pressure differential. However, in the context of endothelial injury or enhanced vascular permeability caused by increased pulmonary arterial pressure, PASMC are exposed to shear stress or to enhanced transmural interstitial flow. Shear stress can increase cytosolic Ca2+ concentration ([Ca2+]cyt). An increase in [Ca2+]cyt in PASMC is a major trigger for vasoconstriction and a key stimulus for cell proliferation, which contribute to vascular remodeling. We show here that shear stress induced increased [Ca2+]cyt in PASMC from patients with IPAH compared with control. Fluid flow shear stress induced enhanced transient and plateau [Ca2+]cyt and larger Ca2+ oscillation amplitude in IPAH PASMC than in control. Expression of TRPM7 and TRPV4 channels, two Ca2+ channels implicated in response to shear stress, both were increased in IPAH PASMC. TRPM7 and TRPV4 blockers 2‐APB and Ruthenium red inhibited flow shear‐induced enhanced [Ca2+]cyt in IPAH PASMC respectively, and the TRPM7 agonist bradykinin induced higher [Ca2+]cyt and [Mg2+]cyto increase in IPAH PASMC than in control, so did TRPV4 agonist 4α□PDD. Knocking down TRPM7 and TRPV4 by siRNA decreased [Ca2+]cyt increase induced by fluid flow. These results demonstrate that fluid flow shear stress induce enhancement of [Ca2+]cyt in IPAH PASMC through the Ca2+ channels TRPM7 and TRPV4, and that mechanical sensitive channels TRPM7 and TRPV4 play a critical role in PAH by modulating [Ca2+]cyt homeostasis.

Grant Funding Source: Supported by NIH R01(HL 066012, HL 115014))
CD4+ T cells contribute to chronic hypoxia‐induced pulmonary hypertension (1090.5)
Authors: Maston Levi , Cannon Judy , Resta Thomas , González Bosc Laura
Published: June 2021

We have previously demonstrated that adaptive immune cells contribute to chronic hypoxia (CH)‐induced pulmonary hypertension (PH). Within the adaptive immune system, CD4+ T cells are required for initiating and maintaining inflammation, suggesting these cells could play an important role in the pathology of hypoxic PH. Therefore, we hypothesized that CD4+ T cells contribute to CH‐induced PH. To test this hypothesis, we isolated CD4+ T cells from lymph nodes of male C57BL/6 wild‐type (WT) mice by negative selection or B cells from spleens of T cell receptor knockout (KO) mice lacking T cells. We then adoptively transferred B cells and CD4+ T cells separately into RAG1 KO mice, which lack mature T and B cells. WT mice and adoptively transferred and sham‐treated RAG1 KO mice were exposed to normoxia or CH (PB=380 mmHg) for 3 weeks, at which point peak right ventricular systolic pressure (RVSP) was measured. As previously observed, sham‐treated RAG1 KO mice were protected from the development of CH‐induced PH as indicated by significantly lower RVSP compared to CH WT mice (p<0.05). In support of our hypothesis, adoptive transfer of CD4+ T cells, but not B cells, to CH RAG1 KO mice restored the PH phenotype to the level of CH WT mice. We conclude that CD4+ T cells are sufficient to drive CH‐induced PH.

Grant Funding Source: Supported by T32 HL07736, R01HL088151, R01 HL088192, R01 AI097202