bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 18: The Role of Matrix Metalloproteinase 12 in Vascular Stiffness, Inflammation and Hypertension
Authors: Pidkovka Nataliya A , Wu Jing , Goldstein Anna E , Thabet Salim , Harrison David G
Published: July 2021

Vascular wall remodeling and inflammation contribute to hypertension. These processes decrease arterial elasticity due to a loss of elastin and deposition of collagen. Metalloproteinase-12 (MMP12) is an elastase produced by macrophages and vascular cells. Cleavage of elastin by MMP12 could increase vascular stiffness and lead to release of pro-inflammatory elastin fragments. We sought to determine if MMP12 contributes to hypertension and vascular stiffness in response to angiotensin II and to understand mechanisms responsible for its expression. To determine if mechanical stretch activates MMP12 we subjected cultured murine endothelial cells to 0%, 5% (normotensive) or 10% (hypertensive) uniaxial stretch for 48 hours. Real-time RT-PCR showed that 10% stretch increases MMP12 mRNA by 4 fold compared to 0% or 5% stretch. Western blots and casein zymography indicated that 10% stretch significantly increases MMP12 protein expression and activity, respectively. Western blots also revealed an increase in elastin fragments in the media of cells exposed to 10% stretch compared to those exposed to 5% stretch. To understand the role of MMP12 in hypertension, we infused angiotensin II in wild-type and MMP12
-/-
mice. In keeping with the studies of cultured endothelial cells, angiotensin II markedly increased vascular MMP12 mRNA (10-fold) and protein levels. The hypertension caused by angiotensin II was markedly blunted in MMP12
-/-
mice (122 ± 3 vs. 173 ± 5, p < 0.0001). Immunohistochemistry with a CD68 specific antibody indicated a significant decrease in macrophages accumulation in the perivascular tissues of MMP12
-/-
compared to WT mice. We also examined distensibility of aortic segments in vitro, and quantified this as the percent change in diameter over a pressure change from 75 to 125 mmHg. Angiotensin II markedly decreased this distensibility index from 24 ± 3 to 9 ± 2%, but only to 16 ± 3% in MMP12
-/-
mice (p < 0.05). In conclusion, these studies show that mechanical stretch in vitro and hypertension in vivo cause a striking increase in endothelial cell and vascular MMP12 expression and activity. MMP12 activation promotes aortic stiffening and ultimately hypertension. Thus, modulation of MMP12 might be a therapeutic target in hypertension.
Abstract 184: Novel Vasoactive Intestinal Peptide-ELP Fusion Protein VPAC-Agonists Trigger Sustained Pulmonary Artery Vaso-Relaxation in Rats with Acute Hypoxia-Induced Pulmonary Hypertension
Authors: Yeh Steve T , Youngblood Bradley L , Georgopoulos Lynne , Arnold Sue , Hamlin Robert L , del Rio Carlos L
Published: July 2021

The natural vasoactive intestinal peptide (VIP) triggers potent pulmonary vasodilatation by activating the G-protein-coupled VAPC receptors, and has been suggested as a therapeutic target in pulmonary artery hypertension (PAH); however, VIP’s clinical utility is limited due to its short half-life. A novel ELP fusion technology (PhaseBio’s) permits the creation of long-acting protein-fusion biopolymer-based VPAC-receptor agonists. Here, the pulmonary/systemic hemodynamic effects of two novel ELP-enhanced VIP analogues (ELP-VIP) were evaluated in anesthetized rats with hypoxia-induced PAH.

Sprague-Dawley rats (259 ± 5 g, n = 25) were anesthetized (propofol), intubated, mechanically-ventilated (95% FiO2), and instrumented for systemic (arterial) and mean pulmonary-artery pressure monitoring. Following instrumentation PAH was induced by acutely decreasing the inspired FiO2 (to ∼10-11%). Subsequently, the animals were assigned to receive either a novel VPAC agonist (VIP-ELP, n = 18: 3-6 mg/kg) or placebo (VEH, n = 11: 0.9% NaCl). Treatments were delivered as acute single-dose boluses given either intravenously (IV) or intratracheally (IT) at a fixed volume.

Reductions in the inspired FiO2 resulted in sustained pulmonary artery pressure increases (+45 ± 3%, 28 ± 1 to 41 ± 1 mmHg; P < 0.05), consistent with hypoxic PAH. Induced-PAH was preserved in the absence of active treatment, as pulmonary pressures remained elevated following placebo administration (VEH: -1 ± 2%, from 40 ± 2 to 40 ± 2 mmHg; n = 11, N.S.). Acute administration of the VIP-ELP analogues resulted in rapid pulmonary artery pressure reductions (-24 ± 3%, from 41 ± 1 to 31 ± 1 mmHg, P < 0.05) with moderate/negligible changes in systemic pressures (MAP: -12 ± 6%) and heart rate (-4 ± 3%). Notably, the pulmonary vaso-relaxation triggered by VIP-ELP agonists was sustained and independent of the route of administration (IV: -17 ± 4%, n = 8; IT: -28 ± 4%, n = 10).

The novel ELP-enhanced VIP analogues triggered rapid and sustained reductions in pulmonary-artery pressures in the setting of induced (hypoxic) pulmonary hypertension (PAH). These effects were independent of the route of administration, as both intravenous/intra-tracheal administrations were efficacious.
Abstract 195: Nitro-Oleic Acid is a Dual Inhibitor of Nox1 and Nox2
Authors: Al Ghouleh Imad , Ranayhossaini Daniel I , Schopfer Francisco J , Freeman Bruce A , Pagano Patrick J
Published: July 2021

Nitrated fatty acids (NFAs) are α,β-unsaturated keto derivatives of ω-3 fatty acids formed from redox reactions of nitric oxide and nitrite and have recently emerged as anti-inflammatory agents that act as electrophiles. NFAs activate Nrf2 and PPARγ signaling and inhibit NF-κB. Recent data suggest a role in inhibition of reactive oxygen species (ROS) production. This function however, remains largely unexplored. The Nox family of oxidases is a major source of ROS implicated in hypertension and other cardiovascular diseases (CVDs). Here we postulate that the NFA nitro-oleic acid (OA-NO
2
) specifically inhibits ROS production by the Nox1 and Nox2 oxidases. Treatment of canonical Nox1-expressing COS-7 cells with OA-NO
2
vs its oleic acid (OA) control resulted in a concentration-dependent inhibition of ROS production (101.5, 98.9, 91.7, 94.8, 54.2, and 45.5 % of vehicle treated groups for 0.1, 1, 2, 4, 8, and 10 μM OA-NO
2
, respectively), with an IC
50
of 6.5 μM. Treatment of phorbol ester (PMA)-stimulated Nox2-expressing COS-7 cells yielded similar results (103.6, 106.6, 112.2, 102.1, 51.8 and 35.1 % vehicle treated groups for the same OA-NO
2
concentration range) with an IC
50
of 7.0 μM. Treatment with the same OA-NO
2
concentrations of Nox4- or Nox5-expressing COS-7 cells did not result in any inhibition. The effects of OA-NO
2
on Nox1 and Nox2 were unique to its electrophilic unsaturated keto structure, as OA (lacking NO
2
group), palmitic acid (lacking both double bond and NO
2
group) and linoleic acid (containing two
cis
double bonds) did not result in any inhibition, demonstrating that fatty acids other than NFAs are incapable of this inhibition. Moreover, experiments using potassium superoxide (O
2
•-
) ruled out any scavenging effects of OA-NO
2
on O
2
•-
. Finally, 10 μM OA-NO
2
inhibited both angiotensin II (AngII)- and PMA-stimulated O
2
•-
in rat aortic smooth muscle cells (4.4 ± 0.9, 10.8 ± 1.7 and 9.0 ± 2.5 in OA-treated vehicle, AngII and PMA groups vs. 5.8 ± 0.6, 5.7 ± 0.3 and 3.3 ± 1.0 pmol O
2
•-
/min/mg protein in OA-NO
2
-treated vehicle, AngII and PMA groups, respectively). Taken together, these results identify NFAs as potent inhibitors of Nox1- and Nox2-derived ROS raising the possibility for their use as therapeutic agents to treat hypertension and other CVDs.
Abstract 198: Oxidative Stress Mediates the Augmented Muscle Mechanoreflex in Peripheral Arterial Disease Patients
Authors: Muller Matthew D , Drew Rachel C , Blaha Cheryl A , Mast Jessica L , Cui Jian , Reed Amy B , Sinoway Lawrence I
Published: July 2021

Background
Exaggerated blood pressure (BP) responses to dynamic exercise predict cardiovascular mortality in peripheral arterial disease (PAD) patients. However, the underlying mechanisms are unclear and no attempt has been made to attenuate this response using antioxidants.

Methods
Three physiological studies were conducted in PAD patients and controls. In Protocol 1, subjects underwent four minutes of low-intensity (0.5-2.0 kg), rhythmic plantar flexion in the supine posture. In Protocol 2, PAD patients received high dose ascorbic acid intravenously prior to exercise. In Protocol 3, involuntary exercise was conducted via electrical stimulation of the tibial nerve. The primary outcome measure was the change in mean arterial pressure (ΔMAP) during the first 20 seconds of exercise (i.e. when mechanoreceptors within skeletal muscle activate the sympathetic nervous system).

Results
Compared to controls, PAD patients had significantly greater ΔMAP during plantar flexion, particularly at 0.5 kg of the most affected leg (11±2 vs. 2±1 mmHg) as well as the least affected leg (7±1 vs. 1±1 mmHg). This augmented response occurred before the onset of claudication pain and was attenuated by ∼50% (i.e. 6 mmHg) in the presence of ascorbic acid. Electrically evoked exercise also elicited larger hemodynamic changes in the PAD patients compared to controls. Further, the ΔMAP during 0.5 kg plantar flexion inversely correlated with the ankle-brachial index, indicating that patients with more severe resting limb ischemia had a larger BP response to exercise.

Conclusions
The BP response to low intensity exercise was enhanced in PAD. Chronic limb ischemia may sensitize muscle afferents and potentiate the autonomic response to muscle contraction in a dose-dependent manner.
Abstract 199: Hydrogen Peroxide Mediates the pH Conditioned Chloride Conductance in Nodose Ganglia Neurons
Authors: Wang Runping , Lu Yongjun , Whiteis Carol A , Benson Christopher J , Chapleau Mark W , Abboud Françs M
Published: July 2021

pH sensitivity has been rarely studied in vagal afferents of nodose ganglia (NG) neurons. Using whole-cell patch-clamp technique in isolated NG neurons, we recently identified a pH-conditioned Cl
-
current (pH-I) that was evoked following 2 or 3 brief (10s) exposures to low extracellular pH (7.0-6.0) in 16 of 22 (70%) cells (FASEB J, 2012). The current is large (904.3±159.9pA) and prolonged, lasting 10∼15 minutes, and causes significant depolarization (Δ35.2±4.4mV). In the present study, we tested the hypothesis that reactive oxygen species (ROS) mediates this pH-conditioned Cl
-
conductance. We found that the rate of increase in fluorescence [(F-F
0
)/F
0
] of NG neurons loaded with dihydroethidine (ROS dye) rose dramatically following the brief exposures to pH 6.0 from a control of 0.04±0.01 to 0.12±0.02 units/min over 10∼15 minutes (n=31 neurons, p<0.01). Moreover superfusion of neurons with H
2
O
2
induced currents that mimicked the pH conditioned currents. Because of similarities between the pH-conditioned Cl
-
conductance and the previously described “swell-activated” Cl
-
current induced with hypoosmotic solutions, we superfused the NG neurons with the H
2
O
2
scavenger PEG-catalase (1000 units/ml). PEG-catalase blocked significantly (p<0.01) the “swell” response to 210 mOsm from 23.3±6.3pA/pF to 0.57±0.39 pA/pF (n=4) as well as the pH-conditioned response from 25.7±6.5pA/pF (n=6) to 6.9±1.4 pA/pF (n=12). The superoxide scavenger PEG-SOD did not affect the current. These results indicate that both pH-conditioned and swell-induced responses are mediated by H
2
O
2
. Opening of these outward Cl
-
conductances that cause sustained depolarization of vagal afferents may induce a beneficial reflex sympathoinhibition during myocardial ischemia/acidosis or initiate a gastro-intestinal post-prandial satiety reflex.
Abstract 206: Hyperleptinemia Increases Blood Pressure and Decreases Pup Weight in Pregnant Rats
Authors: Palei Ana C , George Eric M , Arany Marietta , Cockrell Kathy , Granger Joey P
Published: July 2021

While the relationship of obesity to cardiovascular disease is well recognized, it also has important implications for pregnancy outcomes. Indeed, there is compelling evidence that obesity increases the risk of preeclampsia (PE). The risk of severe and mild PE and PE occurring in early and late gestation are greater in obese and overweight women. Despite the fact that obesity is the leading attributable risk for PE in developed countries, the pathophysiological mechanisms whereby obesity and metabolic factors such as leptin increases the risk for developing PE are unclear. Hyperleptinemia over the levels seen in normal pregnancy has been associated with preeclampsia. Thus the aim of this study was to investigate whether chronic hyperleptinemia causes changes in cardiovascular, metabolic and reproductive systems of pregnant rats. On gestational day (GD) 14, Sprague Dawley rats were assigned to normal pregnant (NP, n=8) group or to NP plus Leptin group (NP+Lep, n=8), in which miniosmotic pump with leptin (0.5 μg/kg/min) was placed intraperitoneally. On GD 19, mean arterial pressure (MAP) was recorded, rats were sacrificed, and blood, placentas and pups were collected. Body weight (BW) and food intake (FI) were measured on GD 16-18. Serum leptin concentration was elevated in NP+Lep compared with NP (0.82 ± 0.05
vs
17.98 ± 2.75 ng/mL, P<0.05). Circulating insulin and glucose levels were similar in NP and NP+Lep groups (both P>0.05). MAP was higher in NP+Lep compared with NP (102.40 ± 2.38
vs
121.30 ± 8.13 mmHg, P<0.05). BW was decreased in NP+Lep compared with NP at GD 19 (330.90 ± 9.08
vs
284.10 ± 8.58 g, P<0.05), probably due to the reduced FI of the NP+Ins group compared with NP during GD 16-18 (23.45 ± 0.61
vs
8.61 ± 0.83 g/day, P<0.05). Although the number of viable fetuses per rat was similar between groups (P>0.05), fetuses and placentas of the NP+Lep group were lighter than those of the NP group (2.29 ± 0.06
vs
2.11 ± 0.06 g and 0.58 ± 0.01
vs
0.50 ± 0.02 g, respectively, both P<0.05). In conclusion, leptin increases blood pressure, despite its effect of reducing body weight during pregnancy, representing a possible mechanism to induce hypertension in preeclampsia. In addition, leptin decreases pup and placental weights, which could lead to abnormal fetal outcomes.
Abstract 21: Leptin-Induced Arterial Pressure Increase: Key Role of the PI3K Pathway
Authors: Harlan Shannon M , Morgan Donald A , Rahmouni Kamal
Published: July 2021

Leptin is an adipocyte-derived hormone that acts in the brain to decrease food intake and increases energy expenditure. Leptin also raises arterial pressure by increasing sympathetic nerve activity (SNA). We previously implicated PI3K as a downstream pathway in leptin receptor (ObRb) signaling that mediates the renal SNA effects of leptin. We used genetic approaches to test the hypothesis that bidirectional changes in PI3K activity (gain vs. loss of function) will be associated with contrasting changes in renal SNA and arterial pressure responses to leptin. PI3K signaling was enhanced in leptin-sensitive neurons in Pten
ΔObRb
mice in which the lipid phosphatase Pten was ablated in cells that express ObRb, leading to accumulating PIP3 in these cells. For loss of function, we used p110α
D933A/WT
mice which carry a heterozygous mutation in the p110α subunit of PI3K specifically disrupting this pathway. Consistent with our previous findings, the increase in renal SNA caused by intracerebralventricular (ICV) injection of leptin (2 μg) was exaggerated and blunted in Pten
ΔObRb
mice (286±41%) and p110α
D933A/WT
mice (-40±6%), respectively, relative to littermate controls (70±4%). These effects were specific to leptin, as the renal SNA response to ICV MTII (melanocortin receptors agonist) was comparable (P=0.3) between controls (196±36%), Pten
ΔObRb
mice (159±98%) and p110α
D933A/WT
mice (194±36%). Interestingly, the Pten
ΔObRb
mice had significantly (P<0.01) higher baseline radiotelemetric mean arterial pressure (MAP, 137±4 mmHg) as compared to littermate controls (107±2 mmHg). In contrast, despite being hyperleptinemic the p110α
D933A/WT
mice had normal (P=0.34) baseline MAP (112±1 mmHg) as compared to controls (113±2 mmHg). We further measured the arterial pressure response to leptin treatment. Intraperitoneal administration of leptin (60 μg) raised MAP in control mice by 13±5 mmHg and this response was significantly (P<0.05) enhanced in the Pten
ΔObRb
mice (+35±14 mmHg). Conversely, the p110α
D933A/WT
mice had a significantly blunted (P<0.05) MAP response to leptin (-3±4 mmHg). Our data demonstrate the importance of PI3K signaling in leptin receptor-containing neurons for the regulation of arterial pressure and in mediating leptin effects.
Abstract 214: (pro) Renin Receptor Stimulates the Expression of Fibrotic Genes in Mouse Collecting Ducts Cells via Wnt/β-catenin Signaling, Independently of Angiotensin II
Authors: Cuevas Catherina A , Gonzalez Alexis A , Inestrosa Nivaldo C , Vio Carlos P , Prieto Minolfa C
Published: July 2021

The prorenin receptor (PRR) is upregulated in the kidney by high angiotensin II (Ang II) states such as those that occur with AngII-dependent hypertension and low salt diet. The PRR is an accessory protein of the vacuolar H-ATPase, which facilitates Wnt/β-catenin signaling. The Wnt/β-catenin pathway is involved in fibrosis processes. In the present study, we aimed to determine whether the stimulation of PRR in mouse collecting duct M-1 cells induces fibrotic genes independently of Ang II, and if this effect is mediated by activation of Wnt/β-catenin. Both Ang II (10
-7
M) and human recombinant prorenin (hRPr; 2,5 x 10
-8
M) treatments (8 and 16 hours) increased mRNA and protein levels of fibronectin and collagen I (1.5±0.08 and 1.5 ± 0.1 fold change, respectibely; p<0.05); however, the effects of hRPr were elicited earlier. Likewise, Ang II and hRPr stimulated the Wnt target genes, cyclin D1 and c-myc (cyclin D1: 2±0.2 for both; c-myc: 1.4 ± 0.03 and 1.2± 0.002 fold change for Ang II and hRPr, respectively; p<0.001). Ang II type 1 receptor (AT1R) blockade with candesartan (10
-7
M) completely prevented the Ang II-dependent stimulation but not the effects of hRPr on Wnt signaling genes. Upregulation of fibronectin and collagen I genes by Ang II or hRP at 16 h was prevented by Wnt signaling inhibition with Pyrvinium Pamoate (10
-7
M). The data indicate that in M-1 cells, activation of AT1R and PRR stimulate the synthesis of fibrotic genes via Wnt signaling by independent mechanisms.
Abstract 223: Genetic Deletion or Pharmacological Blockade of Ddah-1 Impairs Reabsorption of Fluid From the Rat Renal Proximal Tubule Perfused in vivo.
Authors: Bell Tracy , Nie Jingjiang , Tomlinson James , Leiper James , Wilcox Christopher S , Welch William J
Published: July 2021

Nitric oxide (NO) stimulates proximal tubule (PT) Na
+
and fluid reabsorption. Asymmetric dimethylarginine (ADMA) is an endogenous inhibitor of nitric oxide synthase (NOS) but its effects on tubular function are unknown. ADMA is metabolized by dimethylarginine dimethylaminohydrolase (DDAH) whose type 1 isoform is predominant in the PT. SNIPs of DDAH-1 predicted the rate of decline of renal function in patients with chronic kidney disease (CKD). Therefore, we tested the hypothesis that DDAH-1 metabolizes ADMA in the proximal tubule and thereby enhances PT fluid reabsorption (J
v
). J
v
was measured in anesthetized rats by direct in vivo microperfusion and recollection of artificial tubular fluid (ATF) in S2 segments of the PT isolated between oil blocks. Addition of a selective inhibitor of DDAH-1, L-257 (10
-4
M) to ATF did not effect J
v
when compared to addition of vehicle (3.40.6 vs 3.10.3 nl/min/mm). However, L-257 administered as a bolus intravenous injection (60mg/kg) 2hrs before microperfusion of the PT with L-257 significantly reduced J
v
to 1.80.2 nl/min/mm, (P<0.05) and significantly enhanced urine flow rate (2.1±0.3 vs 9.0±1.8 μl/min; P<0.05). Microperfusion of ADMA (10
-4
M) or L-NAME (10
-4
M) into the PT both reduced J
v
significantly to 2.00.2 and 1.80.2 nl/min/mm, respectively (P<0.05). Rats pretreated with a rapid intravenous injection of siRNA targeted to DDAH-1 that reduced its renal mRNA expression significantly also had reduced J
v
(3.1±0.2 vs 2.2±6.2 nl/min/mm; P<0.05) and had a further reduction in J
v
with 10
-4
M ADMA added to ATF to 1.4±0.1 nl/min/mm; (P<0.005). In conclusion, PT fluid reabsorption is regulated by ADMA and its tubular metabolism by DDAH-1. The two hour delay in the effects of a bolus injection of L-257 to diminish J
v
indicated the time required after inhibition of DDAH-1 for accumulation of sufficient tubular ADMA to impair PT reabsorption. Thus, L-257 is a novel regulator of PT function and is a proximal tubule diuretic. SNIPs that alter DDAH-1 in the PT may change renal function and contribute to CKD progression.
Abstract 225: Loss of Either Renal or Systemic ACE2 contributes to the Hypertensive Response to Chronic Angiotensin II
Authors: Ortiz Melo David I , Mattocks Natalie K , Xu Katherine , Griffiths Robert , Coffman Thomas M , Gurley Susan B
Published: July 2021

Angiotensin Converting Enzyme 2 (ACE2) is a monocarboxypeptidase that degrades angiotensin II (AngII). ACE2 is broadly expressed in cardiovascular tissues, with highest expression in the kidney. We have previously shown that mice with targeted deletion of the
Ace2
gene have an exaggerated response to AngII-induced hypertension (HTN) associated with elevated renal AngII levels. Based on these findings, we now hypothesize that ACE2 in the kidney decreases AngII levels and is therefore responsible for its anti-hypertensive effects. To test this, we utilized renal cross-transplantation to eliminate ACE2 specifically from the kidney or all other tissues. We studied male ACE2 knockout (KO) mice and wild-type (WT) littermates on (C57BL/6 x 129/SvEv)F
1
background. Kidney transplants were performed to generate four experimental groups: 1) wild-type, 2) renal ACE2 KO, 3) systemic ACE2 KO, and 4) global ACE2 KO. All kidney function was provided entirely by the single transplanted kidney. After baseline measurements, mice were infused with AngII (1000ng/kg/min) chronically for two weeks while mean arterial blood pressures (MAP) were measured with radiotelemetry. There were no significant differences in baseline MAPs among groups. Similar to our prior study, the response to AngII infusion was exaggerated in mice with global ACE2 deficiency compared to WT control mice (152±6 vs.144±7 mmHg;
p
=0.03, n=10). However, the presence of ACE2 in either the kidney or systemic tissues restored MAPs to levels similar to that of the WT group (renal ACE2 KO 140±7mmHg, p<0.01 vs. global ACE2 KO; systemic ACE2 KO 140±5 mmHg, p<0.01 vs. global KO). This effect was equivalent between the renal KO and systemic KO groups. Global ACE2 deficiency leads to an exaggerated hypertensive response to chronic AngII administration. Thus, restoration of ACE2 in either the kidney or in systemic tissues is sufficient to abrogate this enhanced hypertensive response. Our data suggest equivalent effects of both renal and extra-renal ACE2 in hypertension.
Abstract 226: Elevated Brain MCP-1 Contributes to Neuroinflammation in Hypertension
Authors: Shi Peng , Zhou Guannan , Desland Fiona A , Shan Zhiying , Raizada Mohan K , Sumners Colin
Published: July 2021

Inflammation has been identified as a major contributor to hypertension, and increased inflammation is associated with activation of the renin-angiotensin system (RAS). In this study we hypothesized that the activated RAS increases monocyte attractant protein (MCP)-1, which recruits immune cells (both resident microglia and circulating monocytes) to the paraventricular nucleus (PVN),contributing to the elevated sympathetic tone in hypertension. Treatment of primary neurons cultured from the hypothalamus of newborn SD rats with Ang II or prorenin significantly increased the levels of MCP-1 in the medium (Con: 58±7; Ang II: 191±18; prorenin: 213±23 ng/ml). To determine the effects of MCP-1 on the recruitment of resident microglia, we examined primary microglial migration using a cell migration kit. MCP-1 caused a dose-dependent (10-100 ng/ml) increase in microglial migration (Con: 0±2.9; MCP-1 at 10 ng/ml: 3.7±1.3; 50 ng/ml: 10.7±1.7; 100 ng/ml: 35.2±2.6; unit is arbitrary). In comparison to normotensive WKY rats, SHR exhibited significant increases in blood pressure (148±5 mmHg vs. 98±7 mmHg, n=5 each group). The levels of MCP-1 were dramatically increased in blood serum (77±3.6 vs. 30±0.5ng/ml), cerebrospinal fluid (18±0.9 vs. 3±0.3 ng/ml), and PVN tissues (35±0.5 vs. 4±0.4 ng/ml) of the SHR vs. WKY rats. Bone marrow-derived monocytes dissociated from the same animals were found to express high levels of CCR2 mRNA (4 fold greater in SHR vs. WKY rats), the primary receptor for MCP-1. To examine whether there was an effect of MCP-1 on monocyte recruitment, the ability of monocytes to elicit migration was compared in SHR and WKY rats. The results showed that the migration of WKY monocytes reached a plateau by 50 ng/ml MCP-1. In contrast, SHR monocytes were recruited by MCP-1 in a dose-dependent manner (10-100 ng/ml). At 100 ng/ml, MCP-1 was significantly more effective in recruiting monocytes from SHR than WKY (94±5 vs. 38±6; unit is arbitrary). In summary, these data suggest that RAS components can increase MCP-1 expression, which in turn recruits immune cells such as resident microglia and circulating monocytes. The present study suggests a potential mechanism by which inflammation contributes to hypertension in the presence of RAS activation.
Abstract 246: Challenges in Defining Resistant Hypertension: Prevalence Among Hypertensive Outpatients Varies With Inclusion of Diuretic Use and Medication Dosing
Authors: Hayek Salim S , Deka Anjan , Krishnan Sandeep K , Ortega Legaspi Juan M , DeMoss Benjamin D , Wilmot Kobina A , Abdou Mahmoud H , Patel Aalok D , Kumar Vikas R , Veledar Emir , Devireddy Chandan M
Published: July 2021

Background:
Resistant hypertension (RH) is defined as BP above goal despite the use of 3 antihypertensive agents of different classes, or BP controlled on 4 agents, ideally maximally dosed and including a diuretic. RH studies have used varying interpretations of this definition, with prevalence estimates ranging from 3% to 30%. This study characterizes RH when different definitions are used in the same population.

METHODS:
We calculated the prevalence of RH among consecutive hypertensive outpatients presenting to an academic cardiology clinic over 6 months using 4 increasingly stringent definitions of RH. These included 1) SBP>140 (130 if DM or CKD) on three medications or controlled on four medications regardless of dosage; 2) Definition 1 including a diuretic; 3) SBP>140 (130 if DM or CKD) on three maximally-dosed medications (per JNC-7) or controlled on four medications, at least three which are maximally dosed; and 4) Definition 3 including a diuretic. We compared demographic and clinical characteristics of patients with and without RH using chi-square and t tests.

Results:
Among 1761 hypertensive outpatients, 46 (2.6%), 374 (21.2%), 602 (34.2%), and 739 (42.6%) were using 0, 1, 2, and ≥3 medications, respectively; 856 (47.6%) were using a diuretic-containing regimen. Overall, 500 patients (28.4%) had RH by Definition 1, which decreased to 402 patients (22.8%) using Definition 2 which required a diuretic. However, with inclusion of medication dosage, the number of patients with RH decreased to 29 (1.7%) using Definition 3, and 24 (1.4%) using Definition 4. RH using Definition 1 was associated with African-American race, female sex, coronary disease, diabetes, congestive heart failure, stroke, and end-stage renal disease (p<0.05).

Conclusion:
RH represents a small proportion of the overall hypertensive population when a strict definition of RH including maximal medication dosing and/or the presence of a diuretic is applied. These findings suggest that some patients with RH by more inclusive definitions may benefit from optimization of antihypertensive therapy. Consistent definitions of RH should be applied in the future to ensure that RH is adequately identified and so RH treatment interventions and pathophysiology may be consistently compared.
Abstract 248: Assessing the Long Term Association Between Sodium Intake and Mortality
Authors: Singer Pamela , Cohen Hillel , Alderman Michael
Published: July 2021

Background:
Studies linking sodium intake and mortality have produced conflicting results, with some showing an inverse or J-shaped relationship. An earlier assessment of this population (mean 3.8 years follow-up) revealed an inverse association of sodium to CVD morbidity and mortality. We now report the association with all-cause and cardiovascular mortality during an average 18.4 years of follow-up.

METHODS:
Subjects participated in a worksite hypertension program between 1980-1995. Entry criteria were SBP≥140, DBP≥90, or receiving antihypertensive medications. Sodium intake was assessed with 24-hour urine collection. Antihypertensives were discontinued 3-4 weeks prior to collection, and individuals with BP <140/90 after washout were excluded. Mortality data through August 2009 were obtained from NDI and SS Master Death Files. Unadjusted and adjusted hazard ratios (HR) for sex-specific sodium quartiles with all-cause and CV mortality were calculated using Cox regressions.

Results:
Of 2983 individuals, 66% were male; mean(±sd) age 52.2 (±9.5) years; mean sodium intake 3023 (±1584) mg/day. Mean follow-up time was 18.4 (±5.9) years. There were 878 deaths, including 351 (40%) due to cardiovascular causes. HR and 95% CI (QI vs QIV) for all-cause and CV mortality respectively were 1.24 (1.02, 1.49), p=0.03 and 1.68 (1.20, 2.35), p=0.003 in unadjusted models, and 0.76 (0.61, 0.95), p=0.02, and 0.86 (0.60, 1.25), p=0.44 in adjusted models. Subgroup analysis limited to MI, heart failure and ischemic heart disease also revealed a non-significant direct relationship (p=0.15).

Conclusions:
We observed a direct association between sodium intake and all-cause mortality, and a similar but non-significant trend with cardiovascular mortality. These are in contrast both to unadjusted models and to an earlier study in the same population. Absence of clinical information after 1998, and the gap between sodium determination and follow-up, limits ability to explain these contrasting findings. Studies that obtain prolonged information on dietary intake and clinical experience over time will be required to better assess long-term associations between sodium intake and health outcomes.
Abstract 25: Prorenin Elevates Blood Pressure via the (Pro)renin Receptor: Who Needs Renin When You Have Prorenin in the Brain
Authors: Feng Yumei , Li Wencheng , Peng Hua , Ichihara Atsuhiro
Published: July 2021

Growing evidence supports that the brain renin-angiotensin (Ang) system (RAS) plays an important role in blood pressure (BP) regulation and hypertension. This concept has been perplexed by extremely low renin activity in the brain. We recently reported the presence of prorenin in the mouse brain at a level of 10-fold higher than that of renin. In addition, we and others have identified the localization of the (pro)renin receptor (PRR) in various brain nuclei involved in BP regulation. The binding of prorenin to PRR leads to Ang II formation in vitro. Our hypothesis: Brain prorenin binding to PRR mediates Ang II formation, leading to an increase in BP. To test this, neuron-specific PRR knockout mice (Nefh-PRR) and wildtype (WT) littermates (N=5/group) were each implanted with a telemetric probe for BP recording and an intracerebroventricular (ICV) cannula for infusion of mouse prorenin (100ng/ul), mouse renin (100ng/ul), or Ang II type 1 receptor (AT1R) blocker (losartan, 10ug/ul) at 0.3ul/minute for 10 minutes. Mouse prorenin infusion increased the BP (mmHg) in WT mice (ΔMAP: 41 ± 5); however, the prorenin-induced pressor response was abolished in Nefh-PRR mice (ΔMAP: 5 ± 1). Infusion of mouse renin similarly increased BP in Nefh-PRR (ΔMAP: 27 ± 2) and WT (ΔMAP: 31 ± 5) mice. The pressor response induced by prorenin or renin was completely blocked by the infusion of losartan. The data suggests that ICV prorenin via PRR mediates Ang II-dependent pressor response in WT mice. To determine whether PRR contributes to the development of brain RAS-dependent hypertension, Nefh-PRR and WT littermates (N=8/group) were treated with 50mg of deoxycorticosterone acetate (DOCA) subcutaneously, plus 0.9% NaCl drinking water for 21 days. The baseline BP was similar between Nefh-PRR (101 ± 2) and WT (101 ± 3) mice. BP was increased in WT mice (132 ± 6) by DOCA-salt treatment, while Nefh-PRR mice remained normotensive (108 ± 3). In summary, prorenin via PRR mediates AngII/AT1R-dependent pressor response in the brain. Neuron-specific PRR deletion attenuates the development of DOCA-salt hypertension likely due to the lack of Ang II/AT1R activation. We conclude that prorenin/PRR may be the key factors to initiate the brain RAS and play an essential role in neurogenic hypertension.
Abstract 27: Leptin Reduces Food Intake but Fails to Raise Blood Pressure In Mice With Deficiency of Insulin Receptor Substrate (IRS2) In the Entire Brain or Specifically in Pomc Neurons
Authors: do Carmo Jussara M , da Silva Alexandre A , Hall John E
Published: July 2021

Insulin receptor substrate (IRS2) is one of three major leptin receptor signaling pathways. However, its role in mediating the chronic effects of leptin on appetite, blood pressure and glucose regulation is unclear. In the present study we tested whether deletion of IRS2 in the whole brain or specifically in proopiomelanocortin (Pomc) neurons attenuates the chronic cardiovascular and metabolic responses to leptin. Mice with IRS2 deletion in the entire brain (IRS2
flox/flox
/Nestin-Cre, n=7), specifically in Pomc neurons (IRS2
flox/flox
/Pomc-Cre, n=7) and control IRS2
flox/flox
mice (n=9) were instrumented with telemetry probes for measurement of 24-hr mean arterial pressure (MAP) and heart rate (HR) and venous catheters for saline or leptin infusions. After a 5-day control period, mice received leptin infusion (2 μg/kg/min, iv) for 7 days. Compared to IRS2
flox/flox
, IRS2
flox/flox
/Pomc-Cre mice had similar body weight and food intake (33±1 vs 35±1g and 3.6±0.5 vs 3.8±0.2 g/day) and higher MAP and HR (110±2 vs 102±2 mmHg and 641±9 vs 616±5 bpm). IRS2
flox/flox
/Nestin-Cre mice were heavier (38±1.5 g), had increased food intake (4.5±1.0 g/day), and higher MAP and HR (108±1.5 mmHg and 659±9.0 bpm) compared to control mice. Leptin infusion for 7 days in control IRS2
flox/flox
mice gradually increased MAP by 5 mmHg despite decreasing food intake by 31%. In contrast, leptin infusion did not change MAP in IRS2
flox/flox
/Nestin-Cre and IRS2
flox/flox
/Pomc-Cre mice. The anorexic effect of leptin, however, was not attenuated in IRS2
flox/flox
/Pomc-Cre or IRS2
flox/flox
/Nestin-Cre mice (-35% and -34%, respectively). In addition, deletion of IRS2 in the whole brain or Pomc neurons did not impair the ability of leptin to reduce plasma glucose levels (IRS2
flox/flox
: 170±15 to 130±12; IRS2
flox/flox
/Pomc-Cre: 180±20 to 150±15; IRS2
flox/flox
/Nestin-Cre: 160±10 to 131±20 mg/dL) These results indicate that activation of IRS2 signaling in the CNS, and particularly in Pomc neurons, is essential for the long-term actions of leptin to raise MAP and HR but not for its anorexic or antidiabetic effects. (NHLBI-PO1HL51971/ AHA SDG5680016)
Abstract 279: Involvement of Kcnj5 Mutations on Excessive Aldosterone Sythesis When Incubated With Acth by Cells Isolated From Aldosterone-producing Adenoma.
Authors: Kitamoto Takumi , Suematsu Sachiko , Matsuzawa Yoko , Saito Jun , Omura Masao , Nishikawa Tetsuo
Published: July 2021

Objective:
Our objective was to investigate the steroidogenic activity by in vitro incubating adrenocortical cells, isolated from aldosterone- producing adenomas (APA) tissues with/without mutations of the KCNJ5 gene in order to clarify the mechanisms of autonomous production of aldosterone in patients with primary aldosteronism (PA).

Design and Patients:
We subjected 22 cases with APA and sequenced KCNJ5 cDNA by using resected tissues of APA. We divided them to 16 APAs with mutations of the KCNJ5 gene (mutated group) and 6 APAs without its mutations (wild type group). Isolated cells were prepared soon after removing APA tissues, and the cells were incubated with various stimulants, including ACTH and Bt2cAMP. The mRNA expression of each steroidogenic enzyme was also determined by RT-PCR.

Results:
Responsiveness of aldosterone production (A-P) by isolated cells to ACTH and Bt2cAMP was greater in the mutated group than in the wild type group, although basal amount of A-P incubated for 2hrs without any stimulants was equal between two groups. Responsiveness of A-P to TPA and TPA+ Ca ionophore was greater in the mutated group than in the wild type group. Tissue extracts of mRNA without any stimulations revealed that the mutated group demonstrated higher expression of 17-hydroxylase/3HSD2 than the wild type group, which showed greater expression of CYP11B2 than the mutated group.

Conclusion:
In vitro incubation studies demonstrated that ACTH might play a crucial role on up-regulating autonomous production of A-P in APA tissues with mutations of the KCNJ5 gene, although the basal expression of CYP11B2 was lower in the mutated group. Moreover, the mutated group showed high ratio of the basal expression of 17-hydroxylase/3HSD2, suggesting that the mutated group can produce cortisol rather than aldoterone via supplying 17-hydroxylated substrates at the static condition, while cAMP-dependent systems or C-kinase-dependent systems activated by ACTH seem to strongly promote activation of A-P steps in the mutated group, rather than in the wild type group.
Abstract 280: Modulation of Aldosterone Synthesis in Human Adrenocortical Cells by Estrogens via an Interaction on Beta Estrogen and Gpr30 Receptor Subtypes
Authors: Caroccia Brasilina , Seccia Teresa M , Lenzini Livia , Gonzàz Campos Abril , Kuppusamy Maniselvan , Recarti Chiara , Mareso Sara , Rainey William E , Rossi Gian Paolo
Published: July 2021

Background and aim.
The gender dimorphism in the pressor effect of hyperaldosteronism suggests that estrogens may modulate aldosterone synthesis. Estrogens were suggested to affect adrenocortical cell proliferation via beta estrogen receptor (ERβ), but it remains unknown if they also influence aldosterone synthesis.

Methods.
We therefore investigated the expression of alpha (ERα), ERβ and of G protein-coupled receptor (GPR)30 in HAC15 cells, a human adrenocortical carcinoma cell line. HAC15 cells were stimulated with 10
-7
M 17β-estradiol (E2) alone, or with a selective ERβ antagonist (10
-5
M; Tetrahydrochrysenediol THC), a selective ERα antagonist (10
-5
M; MPP dihydrochloride), a non selective ERα and ERβ antagonist (10
-5
M; ICI 182.780 fulvestrant), a selective GPR30 receptor antagonist (10
-5
M; G-15). The cells were also exposed to the highly selective GPR30 agonist G-1, alone or in the presence of MPP, or THC, or Fulvestrant and G-15. CYP11B2 mRNA expression was measured with quantitative real time RT-PCR (Universal Probe Library Roche).

Results.
The three estrogen receptor subtypes were found to be expressed at different levels (ERβ>>ERα=GPR30) in HAC15 cells. E2 alone or on top of selective ERα antagonism did not alter CYP11B2 expression. At variance, E2 on top of selective ERβ antagonism, or of combined ERβ and ERα antagonism, caused a 5- to 7- fold upregulation of CYP11B2 mRNA, a finding that was replicated by the exposure of cells to G-1 on top of combined ERβ and ERα blockade.

Conclusions
. 1) GPR30 is expressed in human adrenocortical cells at levels that are comparable to ERα, but less abundatly than ERβ subtype receptor. 2) 17β-estradiol activates CYP11B2 synthesis via an interaction between ERβ blockade and GPR30 activation. Hence, by showing a role for estrogens in the regulation of aldosterone synthesis via GPR30 subtype receptor, these results could account for the diverse pressor effects of excess aldosterone in men and in fertile vs post-menopausal women.
Abstract 285: The Ratio Between Heart Period and Diastolic Decay Time Derived From 24-hour Ambulatory Blood Pressure Monitoring is Associated With Cardiovascular Risk.
Authors: Bursztyn Michael , Gavish Benjamin
Published: July 2021

The ratio between the heart period (T) and the time constant (τ), characterizing the arterial pressure decay during the diastole, is a known ventriculo-arterial coupling factor, which is constant in mammals. Previously we have shown T/τ can be determined using standard ambulatory blood pressure(BP) monitoring (ABPM) records and when increased ,predicts all-cause mortality. Here we evaluated the association between increased T/τ and risk-related variation in some continuous clinical and demographic characteristics. We analyzed ABPM records of 2823 hypertensive patients (age 56±16, 54% women, 58% medicated and 9% with diabetes). T/τ was determined using its model expression, as the ratio between T coefficient of variation (SD/mean) and ln(SBP/DBP) variability (SD), where SBP and DBP are the systolic and diastolic BP and T (in sec) is 60/heart rate (HR). One-way ANOVA was applied to T/τ (dependent variable) using as a factor one of the following characteristics after grouping at constant intervals: age [in 5-yr intervals], body mass index (BMI) [5 kg/m
2
], mean SBP, DBP and pulse pressure (PP) [5 mmHg]; the corresponding dipping [5%], and hear rate (HR) [5 bpm]. End groups with size<50 were eliminated. T, τ and T/τ were 0.88±0.13 sec, 1.18±0.48 sec and 0.88±0.42, respectively. T/τ was greater than 0.9, 1.0 and 1.1, respectively, for 37%, 29% and 16% of the patients (1048, 829 and 647 of 2823). T/τ variation with the tested factors was monotonous and highly significant (p<10
-6
for all). T/τ increased with older age, higher BMI, SBP, PP, and lower DBP, HR, and their dipping: T/τ was greater than 1.03 for age>77 yr; >1.06 for BMI>32 kg/m
2
; >1.06 for (mean) SBP>162 mmHg; >1.07 for DBP<68 mmHg; >1.07 for HR<57 beat/min; >1.05, >0.96 and >1.11 for SBP- DBP- and HR dipping smaller than 2.5%, 2.5% and 7.5%, respectively. The finding that T/τ values greater than a relatively narrow threshold (1.05±0.04) corresponds to risk factors level, and tendency tendency of the tested characteristics supports the view that T/τ that deviates from sucha threshold may signify autonomic cardiovascular dysregulation.
Abstract 288: Nocturnal Blood Pressure Elevation or Abnormal Circadian Pattern. Which is Better Associated with Cardiovascular Risk?
Authors: De la Sierra Alejandro , Gorostidi Manuel , Segura Julian , Banegas Jose R , de la Cruz Juan J , Ruilope Luis M
Published: July 2021

Background and aim:
A blunted decrease in nighttime BP with respect to daytime values (the non-dipping pattern) and an absolute increase in nocturnal BP have both been related to a worse cardiovascular prognosis. However, these are not independent variables and little is know about which is better related with cardiovascular risk. The aim of the study was to compare clinical data in untreated hypertensives classified on the basis of both absolute nocturnal BP and the dipping pattern.

Methods:
We included 37096 untreated patients from the Spanish ABPM Registry. They were classified in 4 different groups: Group 1: 15743 (42%) with mean nocturnal systolic BP (NSBP) < 120 mmHg and nocturnal systolic dip (NSD) ≥ 10%; Group 2: 6166 (17%) patients with NSBP < 120 mmHg and NSD < 10%; Group 3: 4847 (13%) patients with NSBP ≥ 120 mmHg and NSD ≥ 10%; and Group 4: 10340 (28%) patients with NSBP ≥ 120 mmHg and NSD < 10%. Groups were compared by ANOVA, Kruskal-Wallis or Chi square statistics (p for trend), with specific comparisons between groups 2 and 3 (Bonferroni corrected t-test, Mann Whitney or Chi square statistics).

Results:
Compared to patients with elevated NSBP but normal NSD, those with abnormal NSD but normal NSBP were more frequently women, diabetics, had reduced eGFR and had more frequently previous history of cardiovascular complications. The worse cardiovascular risk profile was seen in those patients with both elevated NSBP and abnormal NSD (table).

Conclusion:
In untreated hypertensive patients an abnormal dipping pattern is more closely related with diabetes and overt cardiovascular and renal disease than nocturnal BP elevation without circadian pattern alterations
.
Abstract 291: Antihypertensive Effect Of Bia 5-1058 a New Selective Peripheral Dopamine β-hydroxylase Inhibitor
Authors: Igreja Bruno , Pires Nuno M , Wright Lyndon C , Soares-da-Silva Patrío
Published: July 2021

The sympathetic nervous system can alter blood pressure by modulation of cardiac output, peripheral vascular resistance and renal function. One strategy for controlling sympathetic nerve function is to reduce the biosynthesis of norepinephrine (NE) via inhibition of dopamine β-hydroxylase (DβH; EC
1.14.17.1
), the enzyme that catalyses the conversion of dopamine (DA) to NE in sympathetic nerves. BIA 5-1058 is a reversible DβH inhibitor that decreases NE levels in peripheral sympathetically innervated tissues slowing down sympathetic nervous system drive, without effect in brain tissues. In freely moving SHR implanted with radio-telemetry transmitters single administration of BIA 5-1058 showed a dose (3, 30 and 100 mg/Kg) and time dependent effect on blood pressure with no significant effect on heart rate (HR) and total activity monitored over a 96-hour period. The maximum reduction on systolic blood pressure (SBP) was -10.8, -21.1 and -35.2 mmHg for 3, 30 and 100 mg/Kg, respectively and the maximum reduction on diastolic blood pressure (DBP) was -9.9, -18.4 and -24.8 mmHg for 3, 30 and 100 mg/Kg, respectively. The antihypertensive effect of BIA 5-1058 (30 mg/Kg) was further evaluated in combination with efficacious doses of well-known antihypertensive drugs, like the ACE inhibitor captopril, the AT1 receptor antagonist losartan, the diuretic hydrochlorothiazide, beta-blocker metoprolol, the alpha-1 receptor antagonist prazosin, and the calcium channel blocker diltiazem. All drugs were administered orally (single dose) in a cross-over design and the effect was monitored for 72 hours. The combination of BIA 5-1058 with any of the tested antihypertensive drugs caused a stronger and prolonged blood pressure decrease than any of the compounds alone.In conclusion, peripheral DβH inhibitors can be used, alone or in combination with others antihypertensive drugs, to reduce blood pressure.