bluuebunny/crossref_metadata_2025 huggingface dataset:
Differences in phosphorylation‐mediated K+ channel regulation between vascular smooth muscle cells from cremaster and cerebral resistance vessels
Authors: Yang Yan , Braun Andrew P , Davis Michael J , Hill Michael A
Published: June 2021

The activity of maxi K+ channels (BKCa) differs in vascular smooth muscle cells (VSMC) from cremaster muscle (CM) and cerebral (CER) resistance arteries. As BKCa are regulated by cyclic nucleotide‐dependent phosphorylation, we examined channel responses to the cAMP stimulant forskolin, dependence on PKA and PKG, and differences in basal phoshorylation levels. Isolated VSMCs were studied using whole cell patch clamp. Forskolin (10– 30 μM) increased whole cell K+ current in both preparations, increasing current at +70 mV by 170.2 ± 1.2 % in CER VSMCs and 136.1 ± 2.5 % in CM VSMCs. Forskolin‐induced K+ currents showed weak sensitivity to the PKA blocker, KT5720 (0.3 μM), while being abolished by the PKG inhibitor, KT5823 (0.3 μM). Basal phosphorylation was apparent as KT5823 reduced basal K+ current and STOCs in both cell preparations. To further determine contributions from basal phoshorylation, VSMCs were dialyzed with alkaline phosphatase (AP; 350U/ml) via the patch pipette. AP reduced K+ current at +70 mV by 49.3 ± 0.9 % in CER VSMCs compared to 28.6 ± 2.1 % in VSMCs from CM and prevented a further reduction in current by KT5823. The data suggest that K+ conductance can be modulated by cAMP in both preparations by mechanisms involving cross‐talk to PKG. Further, BKCa in CER VSMCs may exhibit a higher level of basal phosphorylation, which contributes to their higher basal activity relative to VSMCs from CM. (Supp NIH HL092241)
Stretch induces CRP3 expression in vein grafts
Authors: Campos Luciene Cristina Gastalho , Miyakawa Ayumi Aurea , Barauna Valério Garrone , Borin Thaiz Ferraz , de Oliveira Dallan Luís Alberto , Krieger José Eduardo
Published: June 2021

Cysteine‐ and glycine‐ rich protein 3 (CRP3) can act as structural protein and potent transcriptional cofactor during muscle differentiation and vascular remodeling. In this context, we have demonstrated, by real time RT–PCR, that CRP3 expression is 10 times higher in smooth muscle cells (SMCs) from human mammary artery vs. saphenous vein (h‐SV) and that CRP3 expression can be induced in arterialized h‐SV using an ex vivo flow through system that mimics arterial condition. The upregulation of CRP3 was primarily dependent on stretch stimulus in SMCs, rather than shear stress in ECs. Here, we assessed the induction and localization of CRP3 in SMCs during arterialization. Using a rat vein arterializations model in vivo, 1–14 days after surgery, CRP3 immunostaining was observed predominantly in the inner layer and 28–90 days it appears more scattered in the vessel. Confocal microscopy and western blot analysis showed that CRP3 is expressed in cytoplasm and nucleous of SMCs under stretch and in early stages of rat arterialization model. Later, the localization was restricted to the cytoplasm. Our data provide evidence that CRP3 is primarily expressed in arterial SMCs but upon stretching there is early expression of CRP3 induction in vein SMC cytoplasm and nucleous. These finding support to the idea that CRP3 may influence vascular adaptation to stress via cytoskeleton rearrangement and gene expression reprogramming.
TLR‐9 activation potentiates the role of ERK1/2 in thromboxane A2‐induced contractions in uterine but not in resistance arteries
Authors: Goulopoulou Styliani , Matsumoto Takayuki , Webb R. Clinton
Published: June 2021

The innate immune system detects viral, bacterial and apoptotic cell‐released DNA using the toll‐like receptor 9 (TLR‐9), which activates the release of pro‐inflammatory cytokines via a signaling cascade involving mitogen‐activated protein kinases (MAPKs). In this study, we investigated the role of TLR‐9 activation in vascular function. We hypothesized that activation of TLR‐9 will increase vascular contractility to thromboxane A2 (TXA2), a potent vasoconstrictor, via MAPK‐related pathways. Mesenteric (MA) and uterine artery (UA) segments from virgin, Sprague‐Dawley rats (12 weeks old) were incubated for 12 hours with a TLR‐9 agonist (ODN 2395, 10−5 M) or vehicle. Concentration response curves to a TXA2 analogue (U46619, 10−9 – 3×10−6 M) were performed in the absence and presence of a p38MAPK inhibitor (SB203058, 10−5 M, 30 min) or an ERK1/2 inhibitor (PD98059, 10− 5 M, 30 min). UA but not MA segments incubated with the TLR‐9 agonist had greater sensitivity to U46619 (UA, TLR‐9 agonist: 6.96 ± 0.09 vs. vehicle: 6.68 ± 0.10). Inhibition of ERK1/2 reduced maximum contraction to U46619 in vehicle‐treated UA and MA by 50% and 40%, respectively (p>0.05). TLR‐9 agonist‐treated MA showed a reduction in U46619‐induced contraction by 50% following ERK1/2 inhibition; however, TLR‐9 agonist treatment did not affect UA responses to U46619. These data suggest that TLR‐9 activation “protects” the role of ERK1/2 in U46619‐induced contractions selectively in UA and not in resistance vessels, imposing vessel‐specific risk for vascular inflammation upon initiation of the innate immune response. (Funded by NIH)
Alteration of CD38 and Two‐Pore Channels expression by hypoxia exposure in Pulmonary Arterial Smooth Muscle
Authors: Lee Suengwon , Jiang Yongliang , Sham James S.K.
Published: June 2021

CD38 is a multifunctional enzyme responsible for the synthesis of cyclic adenosine diphosphated‐ribose (cADPR) and nicotinic acid adenine dinucleotide phosphate (NAADP). cADPR and NAADP trigger Ca2+ release via ryanodine receptors of sarcoplasmic reticulum and NAADP‐sensitive two‐pore channels (TPCs) of lysosomal stores, respectively, and are thought to play important roles in regulation of [Ca2+]i and vascular functions. We recently examined the expression of CD38 and TPC1/2 in various rat arterial smooth muscles. Pulmonary artery (PA) has higher expression of CD38 and TPC1/2 compared to other systemic arteries. However, there is little information on the changes in expression and function of CD38 and TPCs in PA under hypoxia. Here, we used western blot analysis and quantitative real‐time PCR to examine the alteration of expression of CD38 and TPC1/2 in PA smooth muscle cells. Rats were exposed to hypoxic condition (10% O2) up to 1 week in hypoxic chamber. Endothelium‐removed PA tissues were collected in day 0, 1, 2, 3 and 7. Both CD38 protein and mRNA were significantly increased during hypoxia compared to day 0. Likewise, TPC1/2 proteins were significantly elevated after hypoxic exposure. Our results show that expression of CD38 and TPC1/2 in PA are upregulated by prolonged hypoxia; the elevation of their expression may contribute to the alterations in Ca2+ homeostasis in hypoxic pulmonary hypertension.
O2 sensing and Ca2+ release in hypoxic pulmonary vasoconstriction in non‐preconstricted pulmonary arteries from rats
Authors: Aaronson Philip Irving , Connolly Michelle Judith , Prieto-Lloret Jesus , Ward Jeremy P.T.
Published: June 2021

We assessed the roles of Ca2+ release, the electron transport chain and NOX in hypoxic pulmonary vasoconstriction (HPV), the process by which hypoxia constricts pulmonary arteries (PA). HPV in the absence of pretone in 2nd and 3rd order rat PA branches gassed with 95% N2, 5% CO2 for 40 min demonstrated an initial transient tension increase (phase 1) followed by a slow rise in tension (phase 2). Phase 1 was abolished but phase 2 was unaffected by the complex I inhibitor rotenone (1 μM). Both phases of HPV were abolished by the complex IV inhibitor cyanide (10 μM). Pretreatment of PA with superoxide dismutase (200 units/ml), catalase (200 units/ml), their combination, or with the putative NOX inhibitor VAS 2870 (10 μM) had no effect on either phase of HPV. The superoxide scavenger TEMPOL (3mM) reduced phase 1 by 42% and abolished phase 2. 10 μM NED‐19 had no effect on either phase of HPV. The PKC blocker Gö6983 (3 μM) reduced both phases by ~30%. Pretreatment with 100 μM ryanodine reduced both phases 1 by ~70%. Pretreatment with a combination of ryanodine (10 μM) and caffeine (10 mM) to deplete SR Ca2+ abolished both phases. Applying zero Ca2+ ‐PSS ablated phase 1 HPV but had no effect on phase 2, and re‐adding Ca2+ during phase 2 did not increase contraction. These data indicate that O2 sensing mechanisms for phase 1 and 2 differ, but in neither case involve NOX. Phase 2 HPV is apparently fully accounted for by the release of Ca2+ from the SR.
A small molecule inhibitor of signal transducer and activator of transcription 3 (STAT3) protects against angiotensin II‐induced vascular dysfunction and hypertension
Authors: Johnson Andrew William , Faraci Frank
Published: June 2021

Cytokine‐mediated gp130 signaling requires activation of STAT3 in immune cells. Although angiotensin II (Ang II) promotes vascular disease and hypertension in part by formation of proinflammatory cytokines, the role of STAT3 in these processes is unclear. We tested if STAT3 is essential for Ang II‐induced vascular dysfunction and hypertension. Incubation of isolated vessels from normal mice with Ang II (10 nM) increased superoxide (~2 fold) and reduced vasomotor responses to the endothelium‐dependent agonist acetylcholine (Ach) by ~50% vs controls (P<0.05), effects that were prevented by coincubation with a small molecular inhibitor of STAT3, S3I‐201 (10 μM). We also measured systolic blood pressure (BP) in C57BL6 mice infused with saline or Ang II (1.4 mg/kg/day) for 14 days via osmotic minipump, with or without S3I‐201 treatment (5 mg/kg IP, q.o.d.). Ang II increased BP compared to control animals (155±2 and 112±2 mmHg, respectively; P<0.001) and this effect was prevented by S3I‐201(114±3 mmHg). Following systemic treatment with Ang II, dilator responses to Ach were reduced by 30–55% in carotid artery and small cerebral arteries studied in vitro and S3I‐201 treatment prevented most of this impairment (P<0.05). In summary, these findings provide the first evidence that targeting STAT3 with small molecule inhibitors, or other approaches, may protect against vascular disease and hypertension.
Early damage in 2K1C hypertension is driven by cytokine generation of parenchymal cells
Authors: Kaiser Jörg , Cheng Jingfei , Warner Gina M , Knudsen Bruce E , Gray Catherine E , Lien Karen R , Juskewitch Justin E , Grande Joseph P
Published: June 2021

The objective of this study was to define a role for chemokine generation by parenchymal cells versus circulating inflammatory cells in the initiation of renal damage in the 2K1C model of renovascular hypertension. 129 SV mice were subjected to renal artery stenosis (RAS) by placement of a polytetraethylene cuff on the right renal artery. Systolic blood pressure increased within 2 days of surgery. The cuffed kidney developed tubular atrophy, whereas the contralateral kidney developed compensatory enlargement. Immunohistochemical staining of the cuffed kidney revealed less than 2 CD3+ T cells per 200x field, and rare F4/80+ macrophages or CD20+ B cells in the first 2 weeks following RAS, with progressive increases in macrophages and T cells throughout the 6‐week observation period. Despite the relative paucity of infiltrating inflammatory cells, there was a dramatic increase in chemokine generation within the first week following RAS, as assessed by qPCR analysis of renal cortical homogenates, with 25‐fold induction of MCP‐1, 47‐fold induction of RANTES, and 15‐fold induction of MIP‐2. Chemokine generation continued to increase throughout the 6 weeks, during which circulating inflammatory cells infiltrated the cuffed kidney. Increased expression of MCP‐1 was detected in tubular epithelial cells within 1 week of RAS. We conclude that chemokine generation precedes inflammatory cell influx in RAS.
Effect of Hypertension on Dendritic Cells and a potential role of Isoketals
Authors: Kirabo Annet , Chen Wei , Wu Jing , Thabet Salim , Bikineyeva Alfiya , Dikalov Sergey , Roberts Jackson , Amarnath Venkataraman , Davies Sean S , Harrison David Glenn
Published: June 2021

We have previously shown that angiotensin II‐infusion activates dendritic cells (DCs) and that this contributes to blood pressure elevation. Reactive oxygen species (ROS) also contribute to hypertension, but the interaction of hypertensive stimuli, oxidative stress and DC activation are not defined. Isoketals are highly reactive oxidation products of arachidonic acid that cross‐link lysine residues on proteins. These can alter protein function and become antigenic. We sought to determine if hypertension alters DC ROS production and if isoketal formation is involved in hypertension. We found that angiotensin II infusion increased DC superoxide production by >50%. Angiotensin II also caused a massive accumulation of isoketals in various tissues including the heart, kidney and vasculature. In additional experiments, we co‐administered salicylamine, a potent scavenger of isoketals. Salicylamine attenuated development of angiotensin II‐induced hypertension (142.59 ± 8.98 mmHg versus 175.53 ± 5.19 mmHg in control mice). These studies show that hypertension causes oxidative stress in DCs. Moreover, isoketal modified proteins may serve as neoantigens that trigger hypertension.
Metformin treatment of angiotensin II‐hypertensive rat decreases phenylephrine‐mediated increased contraction in pudendal arteries
Authors: Labazi Hicham , Webb R. Clinton
Published: June 2021

Hypertension is a major risk factor for erectile dysfunction (ED). Increased circulating angiotensin II (AngII) levels are associated with ED. Data from our laboratory showed that AngII infusion resulted in increased corpus cavernosum (CC) contractility, resulting in ED. Metformin treatment restored normal CC tone and erectile function in AngII‐infused rats. Pudendal artery (PA), the major artery providing blood flow to the penis, is important for normal erectile function. This study tested the hypothesis that AngII infusion will result in increased PA contractility, thus limiting blood flow to penis, and that metformin treatment will restore normal PA contractility. Male Sprague‐Dawley rats were implanted with mini‐osmotic pumps containing saline or AngII (70 ng/min, 28 days). Animals were then treated with metformin (500mg/Kg/day) or vehicle during the last week of AngII infusion. AngII infused rats displayed ED which was associated with increased phenylephrine‐mediated contraction in PA (% of the U‐46619 contraction) (Emax= 400.7% ± 14.47 in AngII vs Emax= 208.6% ± 7.79 in sham). Metformin treatment restored erectile function in AngII infused animals, and reversed the AngII‐mediated increase in PA contractility (Emax= 277.2% ± 14.55 in AngII+metformin vs Emax= 400.7% ± 14.47 in AngII). We conclude that metfromin treatment may be beneficial to restore normal erectile function.
Proteasome inhibition attenuates angiotensin II‐induced hypertension and vascular remodeling
Authors: Li Shuai , Wang Xuejun , Martin Doug
Published: June 2021

Growing evidence implicates the proteasome in cardiovascular disease. However, its role in hypertension is understudied. Accordingly, this study tested the hypothesis that proteasome inhibition attenuates hypertension development and related vascular remodeling.

Male Sprague Dawley rats were given chronic subcutaneous infusion of angiotensin II (AngII) to induce hypertension or vehicle infusion for 2 weeks. The rats were concurrently treated with a proteasome inhibitor, bortezomib (200 μg/kg 3 times per week). Arterial blood pressure was measured from conscious rats via indwelling catheters. After euthanasia the aorta and tibialis anterior skeletal muscle were collected. Histochemical (Masson's Trichrome) and immunohistochemical (CD31) staining were used to estimate wall to lumen ratio and capillary density respectively.

Compared to vehicle (131±7 mm Hg), Ang II infusion caused a marked increase in arterial pressure (159±4 mm Hg). Aortic wall to lumen ratio was increased (+20%) whereas capillary density in skeletal muscle was reduced (−30%). Concurrent bortezomib treatment markedly attenuated these responses (arterial pressure 138±5 mm Hg, aorta wall to lumen ratio −1%, capillary density −8%).

Collectively, these data suggest that inhibition of the proteasome attenuated Ang II hypertension development and related vascular remodeling. Supported by NIH #2RO1HL63053‐ARRA (DM).
Renal afferent nerve stimulation induces baroreflex resetting through the activation of sympathorenal axis without compromising arterial pressure buffering function
Authors: Tobushi Tomoyuki , Hosokawa Kazuya , Murayama Yoshinori , Saku Keita , Onitsuka Ken , Sakamoto Takafumi , Sakamoto Kazuo , Fujino Takeo , Ikeda Masataka , Kakino Takamori , Ide Tomomi , Sunagawa Kenji
Published: June 2021

Background

Chronic kidney disease is known to induce sympathoexcitation, and nephrectomy of those kidneys results in sympathoinhibition. Recent clinical trials indicated that renal nerve ablation markedly suppressed sympathetic nerve activity (SNA). Since the baroreflex system is a powerful regulator of SNA, we examine how renal afferent innervation impacts on baroreflex function to clarify the pathophysiological significance of sympathorenal axis.

Methods

: In 8 anesthetized rats, we isolated the bilateral carotid sinuses and controlled intra sinus pressure (ISP). We dissected the unilateral renal afferent nerve for electrical stimulation (STM) and measured contralateral splanchnic SNA. We changed ISP from 60 to 180 mmHg stepwise with/without STM and measured arterial pressure (AP) and SNA.

Results

: STM significantly upward‐shifted the sigmoidal ISP‐SNA relationship (ΔSNA=21.2±2.6%, p<0.01) without changing the maximum gain (1.4±0.8 vs. 1.6±0.8, p=NS) or response range (77.9±16 vs. 95.3±52 mmHg, p=NS). The estimated setpoint pressure increased from 188.7±16.3 to 214.2±13.9 mmHg (p<0.05). STM did not change the SNA‐AP relationship.

Conclusion

: STM induces baroreflex resetting through the activation of sympathorenal axis. The fact that the baroreflex gain remains unaltered suggests that the renal deafferentiation by ablation lowers AP without compromising AP buffering function.
The Differential Contribution of Splanchnic and Renal Nerves to the Maintenance of Dahl Salt‐Sensitive (S) Hypertension
Authors: Foss Jason David , Osborn John W , Fink Gregory
Published: June 2021

This study tested the hypothesis that splanchnic nerves play a greater role than renal nerves in the maintenance of elevated blood pressure in the Dahl S rat, a genetic model of salt‐sensitive hypertension. Dahl S rats were fed a low salt diet (0.1% NaCl), instrumented with radiotelemeters to measure mean arterial pressure (MAP) and heart rate (HR), and placed in metabolic cages to measure Na+ and H2O balance. After recovery and a 3 day baseline period, rats were put on a high salt diet (4% NaCl) for 7 weeks. MAP increased ~20 mmHg by the end of the 3rd week. At this time rats underwent either celiac ganglionectomy (CGx; n = 8), renal denervation (RDNx; n = 7), both celiac ganglionectomy and renal denervation (CGx/RDNx; n = 5) or sham surgery (SHAM; n = 7). In all groups, MAP fell after surgery then increased over the remainder of the protocol. However, the magnitude of this increase differed between groups such that 4 weeks post‐surgery, MAP was significantly lower in CGx/RDNx (128 ± 7 mmHg) rats than in CGx (147 ± 6 mmHg), RDNx (150 ± 10 mmHg) and SHAM (165 ± 4 mmHg) rats. The antihypertensive effect of CGx appeared to be masked by a compensatory activation of the renal nerves as evidenced by increases in Na+ and H2O retention. This may explain the additive effect of CGx/RDNx. These data suggest that the splanchnic and renal nerves differentially contribute to the maintenance of Dahl S HTN. Supported by R01HL076312.
Roles for Cytosolic NADPH Redox in Regulating Pulmonary Artery Relaxation by Thiol Oxidation‐Elicited Subunit Dimerization of Protein Kinase G 1α
Authors: Neo Boon Hwa , Kandhi Sharath , Wolin Michael S.
Published: June 2021

The activity of glucose‐6‐phosphate dehydrogenase (G6PD) appears to control a vascular smooth muscle relaxation regulated via cytosolic NADPH oxidation. Since our studies suggest thiol oxidation‐elicited dimerization of protein kinase G 1α (PKG1α) contributes to the relaxation of endothelium‐removed bovine pulmonary arteries (BPA) to H2O2 and responses to hypoxia, we investigated if cytosolic NADPH oxidation promoted relaxation by PKG1α dimerization. Depletion of PKG1α by siRNA inhibited relaxation of BPA to 6‐aminonicotinamide (6‐AN), and attenuated the increase in vasodilator‐stimulated phosphoprotein (VASP) phosphorylation. Relaxation to 6‐AN was not altered by depletion of soluble guanylate cyclase. Depletion of G6PD in BPA with siRNA increased PKG1α dimerization and VASP phosphorylation, and inhibited force generation under hypoxia. Depletion of thioredoxin‐1 and thioredoxin reductase‐1 with siRNA elicited responses similar to G6PD depletion. Thus, cytosolic NADPH redox appears to control relaxation of BPA by PKG1α dimerization through its influence on thioredoxin‐1 redox, and these processes may contribute to vascular responses to hypoxia that are associated with changes in NADPH redox.

Supported by NIH grants HL031069, HL043023 and HL066331
Bosentan and Sildenafil reduce pulmonary arterial hypertension in rats induced by semaxanib and a low oxygen environment
Authors: Senese Peter B. , Huang Jinbao , Janssen Douglas , Neves Liomar A. A. , Gralinski Michael R.
Published: June 2021

Previous work has demonstrated the development of increased pulmonary artery pressures, hypertrophy of the pulmonary arterial vascular smooth muscle, and proliferation of the endothelial vascular lumen in rats following extended exposure to hypoxia and VEGF‐receptor antagonists. We validated this model of pulmonary arterial hypertension (PAH) by evaluating its utility as a clinically relevant paradigm to determine the efficacy of test articles against PAH. Male SD rats (240–370 g) were kept inside a hypoxic tent. Atmospheric oxygen was reduced to 13.5% using an oxygen scrubber. Rats received a single dose of semaxanib (200 mg/kg, s.c.) and were maintained inside the tent for 3 weeks. Rats received oral doses of vehicle (20% cyclodextrin, 10mL/kg once daily), bosentan (BS‐ 300mg/kg once daily) or sildenafil (SL‐ 30mg/kg twice daily). At day 21, pulmonary and systemic arterial pressures and thoracic organ weights were measured. BS and SL‐treated rats exhibited systolic, diastolic and mean pulmonary arterial pressures (21 and 19; 16 and 15; 18 and 17 mmHg, respectively) that were significantly lower compared to the vehicle (30, 22 and 26 mmHg, respectively). There was a trend toward decreased right ventricle to left ventricle weight ratio when comparing BS and SL with the vehicle. There were no significant differences in mean arterial pressure among rats treated with BS and SL as compared to vehicle. In summary, oral administration of BS and SL reduces PAH induced semaxanib and a low oxygen environment in a clinically relevant model.
MicroRNA‐27a and PPARγ regulate endothelin signaling in sickle cell disease‐related pulmonary hypertension
Authors: Kang Bum-Yong , Park Kathy K. , Tan Fang , Sutliff Roy L. , Ofori-Acquah Solomon F. , Hart C. Michael
Published: June 2021

Pulmonary hypertension (PH) is a serious complication of sickle cell disease (SCD) that causes significant morbidity and mortality. The PPARγ ligand rosiglitazone (RSG) attenuates hypoxia‐induced PH and endothelin (ET)‐1 levels. To examine these pathways in SCD, we hypothesized that increased levels of microRNA (miR)‐ 27a reduce PPARγ expression to increase ET‐1 levels and PH. Our results demonstrate that levels of miR‐27a, NF‐κB, and ET‐1 were increased in the lungs of 8–10 week old SCD transgenic mice compared to controls whereas PPARγ levels were reduced. In parallel studies, C57BL/6 mice were exposed to control or hypoxic (10% O2) conditions for 3‐weeks ±RSG (10 mg/kg/d) for the final 10 d of exposure. RSG attenuated hypoxia‐induced increases in miR‐27a in C57BL/6 mouse lung. Human pulmonary artery endothelial cells (HPAECs) were exposed to control (21% O2) or hypoxic (1% O2) conditions for 72 h. Hypoxia increased HPAEC proliferation, miR‐27a, NF‐κB, and ET‐1 expression, and reduced PPARγ levels. These alterations were attenuated by treatment with RSG (10 μM). In contrast, overexpression of miR‐27a or knockdown of PPARγ increased HPAEC proliferation. Collectively, these findings suggest that miR‐27a and PPARγ regulate ET‐1 signaling and that targeting PPARγ may represent a novel therapeutic approach in SCD‐PH pathogenesis. Atlanta VAMC, NIH DK074519/HL102167, Emory/Children Healthcare CEB F16788‐00.
TWIK‐2 potassium channel plays a role in pulmonary arterial hypertension
Authors: Pandit Lavannya M. , Lloyd Eric Edward , Bryan Robert M.
Published: June 2021

Pulmonary hypertension is a major cause of morbidity, for which there remains no cure. Pulmonary vascular tone is regulated by potassium (K+) channels intrinsic to the endothelial and smooth muscle cells in the vessel wall. Although defects in previously identified K+ channels partially explain the pathophysiology of pulmonary hypertension they do not completely address the mechanisms in this disease. The K+ channel, TWIK‐2 is highly expressed in the systemic and pulmonary vasculature. Our laboratory has cloned and constructed a genetic knockout of TWIK‐2. Aortic smooth muscle cells from TWIK‐2 knockout (KO) mice have increased depolarization and hypercontractile aortic segments, leading to systemic hypertension in these TWIK‐2 KO mice. We hypothesized given TWIK‐2 channel's properties in aortic vascular cells, that TWIK‐2 channels play an important role in regulating pulmonary vascular tone. Right sided cardiac catheterization studies showed TWIK‐2 KO mice have elevated pulmonary systolic pressure (38±2 mmHg) compared to wild‐type littermates. (23±2 mmHg). TWIK‐2 KO mice pulmonary vessels reveal smooth muscle hyperplasia compared to WT mice. Ring myography studies reveal that 1st and 2nd order pulmonary arteries from TWIK‐2 KO mice are hypercontractile and respond differently to hypoxia than their wild‐type littermates. We conclude intact TWIK‐2 is important in regulating pulmonary vascular tone.
Combinatorial inhibitory effects on VSV‐GtsO45GFP trafficking in human endothelial cells due to functional haploinsufficiency of BMPRII, eNOS, STAT5a and STAT5b
Authors: Yang Yang-Ming , Lee Jason E , Sehgal Pravin B
Published: June 2021

Lung lesions in idiopathic pulmonary arterial hypertension (PAH) contain enlarged “vacuolated” endothelial and smooth muscle cells with enlarged and fragmented Golgi apparatus and dilated endoplasmic reticulum (ER) cisternae. Familial PAH often involves mutations in or haploinsuffciency of BMPRII but with low penetrance (10–15%) setting up a search for “second‐hit” genes. Recently we observed that acute knockdown of STAT5a or STAT5b or both in HPAECs using siRNAs led to marked Golgi enlargement and fragmentation and cisternal dilatation of the ER. In the present study we used the VSV‐GtsO45GFP ER to Golgi to plasma membrane trafficking assay in EA.hy926 human endothelial cells to quantitatively assess the combinatorial effects of functional haploinsufficiency of BMPRII, eNOS, STAT5a and STAT5b using respective siRNAs. Knockdown of STAT5a, STAT5b, BMPRII, or eNOS one at a time did not significantly affect intracellular trafficking. However, down‐regulation of STAT5a plus STAT5b inhibited VSV‐G trafficking from the ER/Golgi apparatus to the cell surface by 60–70%. STAT5a or STAT5b combined with eNOS siRNA treatments had a similar inhibitory effect. STAT5a or STAT5b combined with BMPRII siRNA also inhibited VSV‐G trafficking. These observations suggest that combinatorial functional haploinsufficiency of BMPRII, eNOS, STAT5a and STAT5b may contribute to intracellular trafficking defects.
Cyclic Nucleotides Cause Divergent Ryanodine Receptor Modulation in Pulmonary Arterial Myocytes from Immature Chronic Hypoxic Sheep
Authors: Rubalcava Monica , Osman Noah J , Blood Quintin , Kim Joon H , Longo Lawrence D , Wilson Sean M
Published: June 2021

Calcium sparks are due to ryanodine receptor (RyR) activation and are intimate to local and global calcium signals in pulmonary arterial (PA) myocytes. RyRs are heavily regulated and cyclic nucleotides, including cAMP and cGMP, can increase RyR activity and thereby augment Ca2+ sparks and global Ca2+ waves. One possibility is that enhanced RyR activity contributes to altered pulmonary arterial reactivity responses found in sheep born at high altitude. Yet, the influence of chronic hypoxia (CH) on cyclic nucleotide regulation of RyRs is unknown. In these studies, we tested the hypothesis that cAMP and cGMP increase Ca2+ sparks and waves in PA myocytes from hypoxic sheep. Spark and wave activity was determined by visual analysis of line‐scan confocal recordings of Fluo‐4 or Fluo‐8 loaded PA from term‐fetal, ~ 10 day old, or adult sheep that lived at 3,200 meters for <100 days. Similar percentages of fetal, newborn, and adult myocytes had sparks and waves before cyclic nucleotide treatment, although newborns had more sparks per recording. cAMP and cGMP increased the number of adult cells with sparks and waves, yet, they respectively decreased the number of newborn and fetal myocytes with sparks. Spark activity was increased in adult myocytes by cGMP. The data suggest that cyclic nucleotides cause divergent regulation of RyRs from PA myocytes of immature CH sheep. NSF MRI 092355 (SMW), NIH P01HD031226, R01HD003807 (LDL)
Glucose‐6‐phosphate dehydrogenase regulates pulmonary artery smooth muscle cell phenotype
Authors: chettimada sukrutha , Gupte Rakhee , Gupte Sachin
Published: June 2021

Hyperplasia and hypertrophy of pulmonary artery smooth muscle cells (PASMCs) are thought to be the leading cause of medial thickening, which obliterate vascular lumen and increase pressure via contributing to hypoxic vascular remodeling. We have previously shown that glucose‐6‐phosphate dehydrogenase (G6PD), the first enzyme of pentose phosphate pathway (PPP), is activated in PASMCs exposed to chronic hypoxia (CH; 72 hrs; 3% O2). In previous studies, we found that the cell numbers determined by CyQuant cell proliferation assay increased by CH. In current study, cell‐proliferation and ‐cycle determined by BrdU assay and flow cytometry, respectively, demonstrate that CH increased both the number of cells in S‐Phase and cell proliferation. Inhibition of G6PD with dehydroepiandrosterone/6‐ aminonicotinamide/siRNA suppressed this increase. Furthermore, we demonstrate that CH evokes de‐differentiation of PASMCs via downregulation of myocardin and SM22α (80% and 31.5% vs. normoxia ), and upregulation of cyclin A and phospho‐histoneH3 (87.3% and 243% vs. normoxia), an S and G2M phase protein, respectively. Intriguingly, G6PD inhibition was able to suppress the de‐differentiation by re‐expressing myocardin and downregulating cyclin D1 and phospho‐histoneH3 expression to normal level. In conclusion, G6PD derived NADPH or the PPP metabolites plays a significant role in regulating PASMCs phenotype.
Modulation of transient receptor potential canonical channel 3 (TRPC3) activity by a novel pharmacological agonist and antagonist in normotensive and hypertensive rats
Authors: Schnackenberg Christine G , Costell Melissa H , Bernard Roberta E , Xu Xiaoping , Washburn David G , Manns Sharada , Marino Joseph P
Published: June 2021

Transient receptor potential canonical channel 3 (TRPC3) is a nonselective cation channel that is associated with hypertension. Previous studies show enhanced basal and angiotensin II‐induced TRPC3 expression in the vasculature of SHR. We tested the hypothesis that TRPC3 plays an important role in the regulation of blood pressure by using a novel TRPC3 agonist and antagonist. In patch‐clamp studies of HEK293 cells over expressing TRPC3, GSK1702934A stimulated basal TRPC3 current (EC50 = 0.06 uM) and GSK2820986A inhibited GPCR‐activated TRPC3 current (IC50 = 0.005 uM). In conscious, chronically instrumented SD rats and SHR, GSK1702934A (1.0 mg/kg, i.v.) rapidly and transiently increased mean arterial pressure (MAP) by 33 ± 3 mmHg and 39 ± 3 mmHg, respectively. GSK2820986A (10, 50, 150 ug/kg/min, i.v.) dose‐dependently blocked the acute pressor response to GSK1702934A (IC50 = 0.009 uM). In telemetered SD and SHR, GSK2820986A (300 mg/kg/d, chow) administration for three days increased plasma GSK2820986A concentrations to 0.02 uM and 0.05 uM, respectively, and had no effect on MAP (100 ± 2 to 105 ± 2 mmHg in SD; 135 ± 2 to 138 ± 2 mmHg in SHR) or heart rate (373 ± 8 to 390 ± 8 bts/min in SD; 322 ± 2 to 330 ± 3 bts/min in SHR). These data suggest that in normotensive and essential hypertensive rats TRPC3 activation acutely increases blood pressure similarly, but TRPC3 has no major role in the maintenance of steady‐state blood pressure.
Activation of Na+/H+ exchanger (NHE) in the macula densa (MD) enhances tubuloglomerular feedback (TGF) in spontaneously hypertensive rats (SHR)
Authors: Fu Yiling , Lu Yan , Ge Ying , Juncos Luis A , Liu Ruisheng
Published: June 2021

NHE stimulates both NO and superoxide production in the MD. We hypothesize activation of NHE enhances NO generation by MD and suppresses TGF in vivo, however, which is impaired in SHR contributing to enhanced TGF response. TGF was determined by measuring maximum changes of stop‐flow pressure, ΔPsf, while increasing tubular perfusion from 0 to 40 nl/min with micropuncture in the absence and presence of a selective NHE inhibitor, 5‐(N,N‐Dimethyl) amiloride (DMA, 10−4M,) in the tubular perfusate in 8–9 wk old WKY and SHR. NHE inhibition augmented TGF in WKY (ΔPsf 9.69±0.56 v.s. 12.53±0.57 mmHg, vehicle v.s. DMA, p<0.01), while showed no effect in SHR (10.94 ±1.01 v.s. 11.89±1.03 mmHg). In the presence of L‐NMA (10−3M, a NOS inhibitor), NHE inhibition with DMA further enhanced TGF in WKY (12.83±2.04 v.s. 16.63±1.85 mmHg, L‐NMA v.s. L‐NMA plus DMA, p=0.01), while it blunted TGF in SHR (12.45±1.07 v.s. 9.97±0.64 mmHg, p=0.01). In isolated microperfused rabbit juxtaglomerular apparatus, the net NO generation by the MD increased by 287.1% ± 22.1 when switching perfusion solution from 10mM to 80 mM NaCl in the presence of tempol (10−4M) to scavenger superoxide, while it increased only 32.3% ± 1.9 when we repeated the switch and blocked NHE with DMA (p<0.01). We conclude that activation of NHE blunts TGF via increasing NO generation by the MD, while this mechanism is impaired in SHR, probably due to the reduced NO bioavailability.
NARROW CONGENICS REVEAL MULTIPLE GENDER‐SPECIFIC BLOOD PRESSURE LOCI AROUND THE RENIN GENE
Authors: Quinn Carol Patricia Moreno , Hoffman Mathew J , Flister Michael J , Endres Bradley , Greene Andrew S , Lazar Jozef , Jacob Howard J. , Xiao Bing
Published: June 2021

Recent studies in humans and animal models suggest that hypertension is determined by different genes in male and females. The Dahl Salt‐Sensitive hypertensive (SS) rat develops salt‐sensitive hypertension and substitution of Chr 13 from the normotensive Brown Norway (BN) rat onto the SS genetic background (SS.13BN consomic), attenuates the development of salt‐sensitive hypertension in both males and females, and restores renin levels and vasodilatory function. Introgression of a 4Mbp BN segment harboring the renin gene attenuated the development of salt‐sensitive hypertension in female but not in male SS rats, suggesting a sex‐specific effect of this gene on blood pressure. In order to narrow the region responsible for renin regulation and blood pressure we developed sequentially narrow overlapping subcongenic strains for this region. We measured blood pressure in these subcongenic strains, and we found two blood pressure loci located within 1 Mbp in females, with a 30 mmHg effect on blood pressure, 50 kb below the renin gene. We also found a locus for blood pressure in males 1 Mbp below renin, with a 20 mmHg effect on blood pressure. The data suggests that there are a different set of genes that regulate blood pressure in males and females in the renin region. By sequence and expression analysis of the candidate regions, we identified all sequence variants and prioritized the candidate genes for hypertension. HL‐82798
Weight loss surgery decreases platelet activation in morbidly obese patients
Authors: Dobrydneva Yuliya , Periasamy Monica , Galkina Elena , Kuhn Norine , Lieb David , Fontana Mark , Wohlgemuth Stephen , Nadler Jerry
Published: June 2021

Bariatric surgery is the most effective treatment to alleviate morbid obesity and its comorbidities, such as type 2 diabetes. Platelets are crucial for the development of thrombosis that often accompanies obesity and diabetes. Hypothesis: bariatric surgery attenuates platelet activation therefore decreasing thrombosis risk. Objective: to determine the effect of bariatric surgery on platelet function in morbidly obese diabetics and non‐diabetics, and compare two types of surgery, Roux‐en‐Y Gastric Bypass (RY) and Lap Banding (LB). Prothrombotic platelet‐monocyte aggregates (PMA) and P‐selectin/PAC‐1 expression on platelets were evaluated pre‐and post‐surgically in whole blood by flow cytometry. In morbidly obese patients, PMA are drastically elevated compared to non‐obese controls. Bariatric surgery decreased platelet activation in all patients. PMA levels (baseline and ADP‐stimulated) decreased early at 1 month after surgery. More significant decrease was in diabetics and after RY. P‐selectin/PAC‐1 expression in ADP‐stimulated blood decreased later at 3 months after surgery. There is no correlation between weight loss and decreased platelet activation after RY, but they do correlate after LB. Therefore gastric bypass appears to be more rapid and efficacious than gastric banding in reducing platelet hyperactivity and alleviating thrombosis risk associated with morbid obesity.
EFFECT OF EXOGENOUSLY ADMINISTERED H2S ON THE CARDIAC AND RENAL FUNCTION IN HYPERTENSIVE DIABETIC RATS
Authors: Rathore Hassaan Anwer , Din Fiazud , Abdullah Mohammad H , Khan Ayaz A , Zubaid Munavvar , Abdullah Nor Azizan , Johns Edward J
Published: June 2021

This study investigated the action of exogenously administered H2S on mean arterial pressure (MAP), heart rate (HR) and renal function in combined states of hypertension and diabetes.

Groups (n=6) of spontaneously hypertensive rats (SHR) or DOCA salt hypertensive (DOCA) rats received vehicle, streptozotocin (STZ) or STZ plus Sodium Hydrogen Sulphide (NaHS), at 56μmol/Kg i.p daily for 35 days; theerafter, MAP, HR and GFR were measured in Ketamine + Xylazine anaesthetized rats. Data, mean±SEM, were subjected to ANOVA and significance set at p<0.05.

MAP of SHR‐STZ was higher than DOCA‐STZ (145±2 vs 155±2) (p<0.05) whereas it was similar in the SHR‐STZ‐NaHS (132±3 mmHg) and DOCA‐STZ‐NaHS groups. HR of SHR‐STZ at 343±5 b/minwas higher than DOCA‐STZ 314±7b/min (p<0.05) but was similar (312±19b/min) in SHR‐STZ‐NaHS vs DOCA‐STZ‐NaHS. GFR of DOCA‐STZ was lower than SHR‐STZ (0.5±0.03 vs 1.1±0.1ml/min/100g) whereas SHR‐STZ‐NaHS was 66% higher than that of DOCA‐STZ‐NaHS (p<0.05).

Improved cardiovascular function with NaHS was likely due to decreased HR and reduced renovascular resistance through vasodilation in both DOCA and SHR concomitant with the development of diabetes.
Palmitic acid activates murine mast cells via Toll like receptor 4 (TLR4)
Authors: Zhang Hanying , Zhao Junxing , Wang Hui , Du Min , Zhu Mei-Jun
Published: June 2021

Obesity has been closely correlated with aberrant immune status and increasing risk for associated inflammatory diseases. Free fatty acid, which increases in obese condition, is demonstrated to be ligands for Toll like receptor (TLR) 4. Mast cells are critical mediators of innate immunity.

Objective

To explore the role of TLR4 in mast cell activation under obese conditions.

Methods

P815 mast cells and primary bone marrow mast cells (BMMC) isolated from wild‐type and TLR4 knockout (KO) mice were treated with palmitic acid (PA), and mast cell degranulation, proliferation and associated signaling were analyzed.

Results

PA treatment increased tryptase release and inflammatory cytokine expression in P815 cells; TLR4 inhibitor TAK‐242 blocked this activation but didn't affect their proliferation and apoptosis. Consistently, BMMC separated from TLR4 KO mice failed to be activated by PA despite activation of wild‐type BMMC.

Conclusion

TLR4 plays important roles in mediating PA induced mast cell activation. Due to the importance of mast cells in inflammation and innate immunity, our data demonstrate that PA induces activation of mast cells via TLR4, which may provide a novel mechanism explaining low‐grade inflammation induced by obesity (RR016474; USDA‐NRI 2008‐35203‐19084; 2009‐65203‐05716).
Regional body volumes in nonobese and obese women and men
Authors: Babb Tony G , Haller Sarah F , Bernhardt Vipa , Locke Kenneth W
Published: June 2021

Although regional fat distribution is remarkably similar between nonobese and obese women and men, it is unknown whether the compartment volumes are similar between genders or equally increased in obese women and men.

We examined abdominal volumes (AVs) in 11 nonobese women (30 yr, 21 BMI), 9 nonobese men (30 yr, 23 BMI), 10 obese women (33 yr, 37 BMI), and 10 obese men (36 yr, 35 BMI). AVs were calculated from whole body MRI and analyzed by independent t‐test with Bonferroni correction (p<0.0125).

Subcutaneous (subQ) AV occupied 23±4% and 20±5% of outer AV (p>0.05) in nonobese women and men, respectively, and 53±4% and 37±8% in obese women and men, respectively (p<0.0001). Anterior and posterior subQ AVs did not differ between nonobese women and men (p>0.05); but were greater (p<0.0002) in obese women than obese men. Inner and outer AVs were smaller (p<0.001) in nonobese women than men; while in obese women only inner AV was smaller than in men (p<0.0001). All AVs were greater (p<0.0001) in obese than nonobese women except for inner AV (p>0.0125), and all were greater (p<0.0001) in obese than nonobese men.

Thus, despite gender differences in outer and inner AVs, anterior and posterior subQ AVs are similar between nonobese women and men. The increase in AV in obese women is mostly due to an increase in subQ, but not inner AV; while in obese men it is due to increases in both subQ and inner AV.

Lithera, Inc, HL096782, King's Fund, ALA, AHA.
The role of beta 1 adrenergic receptor in Non‐Alcoholic Fat Liver Disease development
Authors: Fernandes Gustavo Werpel , Marcelino Cícera Pimenta , Ribeiro Miriam Oliveira , Lancelotti Carmen Lucia , Brum Patricia Chakur , Passarelli Marisa
Published: June 2021

Non‐Alcoholic Fat Liver Disease (NAFLD) is characterized by the development of macrovesicular steatosis in the absence of significant consumption of alcohol, which can progress to nonalcoholic steato‐hepatitis (NASH). β1 adrenergic receptor may be related to development of NAFLD, since knockout mice for the β1adrenergic receptor (β1KO) are obese when placed on high fat diet. Thus, the aim of this study is to determine the role of β1 adrenergic receptor in NAFLD development. For that we treated β1KO with high fat diet (40%) for 8 weeks. The β1KO mice were significantly more obese than the WT animals (21.8±3.4g β1KO vs. 17.4±3.37g WT, p<0.05) and have higher white adipocyte area (10.8±0.34 β1KO; 6.20±2.06 WT; p<0.001). The liver of β1KO mice showed severe steatosis and fibrosis. The expression of LDL‐R protein was increase in β1KO when compared to WT treated with high fat diet (1307±130 β1KO vs. 627±33 WT, p<0.05). The expression of MTTP protein in β1KO was significantly reduced (568±6 β1KO vs. 837±22 WT; p<0.05). In conclusion, the results showed that the β1 adrenergic receptor are involved in lipid metabolism in liver and has direct influence on expression of proteins related to lipid metabolism.
Characteristics and Hypertensive Actions of Renal Medullary NALP3 Inflammasomes in Mice
Authors: Xia Min , Xiong Jing , Boini Krishna , Abais Justine , Li Pin-Lan
Published: June 2021

Recent studies have indicated that local inflammation is importantly involved in the regulation of renal function and arterial blood pressure. However, it remains unknown how such local inflammation is triggered intracellularly in the kidney. The present study was designed to characterize the expression and function of NALP3 inflammasome in the kidney and also test its role in renal medulla of arterial blood pressure regulation. IHC analysis showed that NALP3, ASC and caspase‐1 were universally distributed in the kidney. Biochemical analysis showed that caspase‐1 activity and IL‐1β production was 2 fold higher in the renal medulla than in cortex. For renal nephron segment analysis, confocal microscopy and RT‐PCR analysis revealed that NALP3, ASC and caspase‐1 were enriched in thick ascending limb of Henle. In chronic animal experiments, medullary interstitial infusion of ATP (1 mM/100 g bw./hr) significantly increased the salt sensitivity of arterial blood pressure (from 100±2 to 123±2 mmHg). Pretreatment of mice with WEHD, an caspase‐1 inhibitor (0.1 mg/100 g bw./d) blocked the ATP‐induced hypertension (100 ±4 mmHg). The sodium balance studies showed that ATP increased sodium retention by 69%, but not in WEHD group. Confocal microscopy showed that increased colocalization of NALP3 with ASC or caspase‐1 in ATP infused mice compared to control mice fed a high salt diet. Consistently, caspase‐1 activity (1.4 fold) and IL‐1β production (1.4 fold) increased in the renal medulla in ATP‐infused mice versus vehicle‐infused mice. IHC analysis of CD43 (a T‐cell marker) showed that T‐cells were accumulated more in the ATP‐infused renal medulla. In conclusion, the NALP3 inflammasome molecules are most abundant in the kidney and that activation of NALP3 inflammasomes in the renal function increases the salt sensitivity of arterial blood pressure resulting in sustained hypertension (Supported by NIH grants HL091464, HL75316, DK54927).
Female Spontaneously Hypertensive Rats (SHR) Have Higher Expression of TGF‐β and Smad Signaling in Mesenteric Arteries Following the Development of Hypertension
Authors: Tipton Ashlee Joy , Sullivan Jennifer C.
Published: June 2021

TGF‐β contributes to vascular damage in hypertension. Our lab has shown that female SHR have higher TGF‐β excretion than male SHR. This study tested the hypotheses that 1) hypertensive (HT) females have greater vascular TGF‐β expression than males and 2) females have greater increases in TGF‐β with the development of hypertension. Aorta and mesenteric arteries were isolated from HT (13 wks) female and male SHR and protein expression was measured. HT females had greater TGF‐β expression than males in mesenteric arteries (densitometric units (DU): 0.5±0.09 vs 0.2±0.03, p<0.5). TGF‐β levels were comparable in aorta of HT females and males. To determine if the sex difference in TGF‐β was dependent on blood pressure, mesenteric arteries were isolated from pre‐HT (5 wks) SHR. TGF‐β expression was comparable in pre‐HT females and males (DU: 0.1±0.02 vs 0.1±0.03, NS), indicating an increase in TGF‐β with hypertension only in females (p<0.05). TGF‐β effects are mediated by smad2/3. HT females had greater smad2/3 expression in mesenteric arteries than males (DU: 0.7±0.1 vs 0.4±0.07, p<0.05), and smad2/3 expression increased with hypertension only in females (DU: 4.3±0.6 vs 2.6±0.4, p<0.5). In conclusion, the development of hypertension is associated with increased TGF‐β expression and signaling in small arteries from female, but not male SHR; suggesting a greater role for TGF‐β in the development of hypertension in females.