bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract P423: Chronic Doxycycline Administration Inhibits Angiotensin Converting Enzyme Activity and Exerts Antioxidants Effects in Renovascular Hypertensive Rats
Authors: Rizzi Sanchez Elen , F Bonacio Giselle , A Guimaraes Danielle , Oliveira Paula Gustavo , Henrich Amaral Jefferson , Becker M Fernandez Victoria , Tanus-Santos Jose Eduardo
Published: July 2021

Doxycycline (Dox), an established matrix metalloproteinase (MMP) inhibitor, and angiotensin-converting enzyme inhibitors (ACEi) present beneficial cardiovascular effects which could be consequence of their antioxidants properties. Furthermore, some evidences have shown other biochemical similarities between Dox and ACEi suggesting that Dox could also inhibit ACE and ACEi directly interact with MMPs and inhibit these proteases. Supporting this idea, Dox is able to chelate divalent metals, such as calcium and zinc, which are essential for the ACE activity. In this regard, we hypothesized that Dox inhibits ACE activity in hypertensive rats which leads to a reduction in ROS production and decrease hypertension. Sham-operated or 2K1C hypertensive rats were treated with Dox (30 mg/Kg/day) or water for 4 weeks. Systolic blood pressure (SBP) was monitored weekly. Dox treatment reduced SBP in 2K1C rats from 200±12 mmHg to 158±8 mmHg (P<0.05). In addition, Dox treatment attenuated reactive oxygen species (ROS) levels in hypertensive animals measured by lucigenin chemiluminescense (in RLU/mg of aorta: from 711±1.0 to 377±93; P<0.05). ACE activity in aorta was increased in untreated 2K1C rats (22±0.8 nmols/min/g) when compared with the Sham group (12±1.0 nmols/min/g; P<0.05) and Dox treatment was able to reduce ACE activity in 2K1C rats (15±2.0 nmols/min/g; P<0.05). To evaluate whether Dox inhibits ACE activity
in vitro
, aortic tissues from 2K1C rats were incubated with Dox or Captopril (a known ACEi ). Dox
in vitro
did not affect ACE activity while captopril inhibited 80% of its activity. To verify whether Dox could inhibit ACE activity
in vivo
, an acute assay was performed with different doses of angiotensin I (in μg/Kg: 0.03, 0.3 and 3), after the single administration of Dox or saline (i.p.). Angiotensin I infusion increased mean arterial pressure dose-dependently and Dox pretreatment did not attenuated these increases significantly (P>0.05) as Captopril.Taken together, these results show that chronic treatment with Dox inhibits ACE activity in aorta of 2K1C rats, which contribute to ROS reduction and SBP attenuation. However, the mechanism by which Dox inhibits ACE activity needs further investigation.
Abstract P435: Differential Responses of Cerebral Cortical and Renal Cortical Microvessels to Perfusion Pressure and Angiotensin II: Effect of Angiotensin II or DOCA/salt Hypertension
Authors: Li Lingli , Lai En Yin , Welch William J , Wilcox Christopher S
Published: July 2021

Background:
The brain and kidney autoregulate their blood flow well yet both suffer from hypertensive damage. We found that a pressor infusion of angiotensin II (Ang II) reduced renal blood flow yet did not change cerebral blood flow. Therefore, we tested the hypothesis that their myogenic and Ang II responses differed.

Methods:
Cerebral cortical microvessels (cerebral) and renal afferent arterioles (afferent) were isolated and perfused from mice after 4 weeks of hypertension from Ang II infusion /high salt/uninephrectomy (Ang II hypertension) or DOCA/high salt/uninephrectomy (DOCA/salt hypertension) or normotensive controls without Ang II or DOCA (n=4-6 per group).

Results:
Normal cerebral and afferents had similar myogenic responses (Δ diameter: cerebral -21±3 versus afferent-19±2%, NS), but bath addition of Ang II or norepinephrine contracted afferents strongly (Ang II: -48±5%, P<0.001, NE: -95±2%, P<0.001), yet cerebrals were entirely unresponsive. Myogenic responses in Ang II hypertension were reduced selectively by 40% in cerebral microvessels compared to controls (-13±3 versus -21±3%, P<0.001) yet maintained in afferents (-17±3 versus -19±2%, NS). However, myogenic responses in DOCA/salt hypertension were maintained in both groups. Contractions to Ang II in cerebral microvessels were increased in Ang II hypertension (-5±2 versus 0±1%, P<0.01) and increased in DOCA/salt hypertension (-18±8 versus -2±2%, P<0.01). In contrast, contractions to Ang II in afferent arterioles were reduced 50% in Ang II hypertension (-23±5 versus -48±5%, P<0.001) and reduced 25% in DOCA/salt hypertension (-38±6 versus -50±10%, P=0.05).

Conclusions:
The kidney is well protected from hypertension and excessive Ang II vasoconstriction. However, the breakdown of myogenic responses in the cerebral microvessels during Ang II hypertension and the enhanced Ang II responses in the cerebral microvessels during Ang II and DOCA/salt hypertension make the brain especially vulnerable to hypertensive ischemia or damage.
Abstract P442: Estradiol Treatment Attenuates the Ovariectomy-induced Increase in Blood Pressure in the Middle Aged Dahl Salt Sensitive Rat as Long as Treatment is Initiated Immediately After Ovariectomy
Authors: Ji Hong , West Crystal A , Wu Xie , Pai Amrita , Souza Aline , Kadam Parnika , West David A , Zhu Michelle J , Sandberg Kathryn
Published: July 2021

Introduction:
Several studies have shown that ovariectomy increases blood pressure and that 17beta-estradiol (

E
2

) treatment can attenuate this effect; however, the majority of these studies were conducted in young animals in which E
2
replacement was initiated soon after ovariectomy. Since most women who experience ovarian hormone loss are middle aged rather than young, this study investigated the effect of E
2
replacement in middle aged Dahl salt-sensitive (
DS
) rats. In addition, the time at which E
2
replacement was initiated was examined.

Methods:
DS rats were ovariectomized (
OVX
) at 4.5 months (
mo
) (

DS-OVX
Y

) and 7mo (

DS-OVX
O

). At 7mo, half of OVX DS rats in each group were treated with E
2
for 4 weeks (

DS-OVX
Y
+E
2
, DS-OVX
O
+E
2

). Mean arterial pressure (
MAP
) was measured by telemetry in all treatment groups from 7-8mo. Body weights were measured throughout the experiment and plasma angiotensin II (
AngII
) was determined at time of sacrifice (8mo) by RAS fingerprint.

Results:
E
2
treatment at 7mo had no effect on MAP in the DS rats OVX at early age [MAP (mmHg) at 8mo: DS-OVX
Y
, 180±5.3 vs. DS-OVX
Y
+E
2
, 179±4.6, ns; n=4/group]. In contrast, one month of E
2
treatment prevented the ovariectomy-induced increase in MAP when OVX at late age [MAP (mmHg): DS-OVX
O
, 191±1.6 vs. DS-OVX
O
+E
2
, 177±2.9, p<0.0001 (Two-way ANOVA); n=4/group]. E
2
reduced the body weight (
BW
) by 26g in the DS-OVX
Y
group and prevented the ovariectomy-induced gain in BW by 39g in the DS-OVX
O
rats [BW(g): DS-OVX
Y
, 349±9 vs. DS-OVX
Y
+E
2
323±7, p<0.05; DS-OVX
O
, 334±7 vs. DS-OVX
O
+E
2
, 295±6, p<0.01; n=7-9/group]. E
2
also reduced plasma AngII in both the DS-OVX
Y
and DS-OVX
O
groups to similar extents [AngII (pg/ml): DS-OVX
Y
, 112±19 vs. DS-OVX
Y
+E
2
, 68.0±7.8, p<0.05; DS-OVX
O
, 113±11 vs. DS-OVX
O
+E
2
, 63.9±6.1, p<0.05; n=7-9/group].

Conclusions:
These findings suggest E
2
replacement can attenuate the increase in MAP in middle age as a result of ovarian hormone loss. This study also supports the timing hypothesis that suggests E
2
treatment loses its protective blood pressure lowering effects if there is a significant time delay between ovarian hormone loss and E
2
replacement. These findings have implications for women who are ovarian hormone deficient and are considering E
2
replacement therapy.
Abstract P446: Sestrin2 Suppresses Age-related Hypertrophy by Inhibiting mTORC1 Signaling Pathway
Authors: Quan Nanhu , Cates Courtney , Rousselle Thomas , Li Ji
Published: July 2021

Introduction:
The mechanistic target of rapamycin complex 1 (mTORC1) plays a critical role in the regulation of cell growth and energy state. A novel stress-inducible protein, Sestrin2 was recognized as a sensor for mTORC1 pathway.

Hypothesis:
The cardiac mTORC1 activation modulated by Sestrin2 is impaired in aging that sensitizes heart to hypertrophy.

Methods:
C57BL/6J young WT (4-6 months) and aged WT mice (24-26 months), and young Sestrin2 knockout mice (4-6 months) were subjected to transverse aortic constriction (TAC) for pressure overload. The
ex vivo
working heart perfusion was used for measuring substrate metabolism.

Results:
The protein levels of cardiac Sestrin2 were decreased with aging. There are no phenotypic differences in young WT, aged WT and Sesn2 KO mice under normal physiology, while aged WT and Sesn2 KO
versus
young WT mice exhibit bigger hearts after 4 weeks of TAC surgery. The echocardiography showed an impaired cardiac function of aged WT and Sesn2 KO hearts by pressure overload. The pressure overload-induced phosphorylation of mTOR and mTORC1 downstream effectors 4E-BP1 and p70S6K were augmented in aged WT and Sesn2 KO
versus
young WT hearts. The swollen mitochondria with severely disrupted cristae and higher levels of redox markers pShc
66
and 4-hydroxynonenal were observed in aged WT and Sesn2 KO
versus
young WT hearts by pressure overload. The rate of glucose oxidation and fatty acid oxidation were impaired in the aged WT and Sesn2 KO
versus
young WT hearts by pressure overload. Intriguingly, pressure overload induced an interaction between Sestrin2 and GATOR2, a complex of unknown function that positively regulates mTORC1. Moreover, the binding affinity between Sestrin2 and GATOR2 is impaired in the aged WT hearts (p<0.05
vs.
young WT). Furthermore, Adeno-associated virus 9 (AAV9)-Sestrin2 were delivered into the aged WT and Sesn2 KO hearts
via
a coronary delivery approach that rescued the protein levels of Sestrin2, attenuated mTORC1 activation and increased the tolerance of both aged WT and Sesn2 KO hearts to pressure overload.

Conclusions:
Cardiac Sestrin2 is a sensor for mTORC1 pathway in response to pressure overload. Sestrin2 deficiency in aging could be a reason for an increased sensitivity to hypertrophy in the elderly.
Abstract P452: Evaluation of Pathological Basis of a Shrsp-based Congenic Strain Characterized by Early Stroke Occurrence
Authors: Ohara Hiroki , Ngarashi Davis L , Nabika Toru
Published: July 2021

Objectives:
The stroke-prone spontaneously hypertensive rat (SHRSP) is a well-known model for essential hypertension and cerebral stroke. In the previous studies, we have identified a major blood pressure (BP) QTL on chromosome (chr) 1 of SHRSP and have explored gene(s) on the BP QTL responsible for hypertensive phenotypes of this strain through physiological analyses using reciprocal congenic strains between SHRSP and a normotensive control (Wistar-Kyoto rat, WKY). In a recent observation, we unexpectedly found that a SHRSP-based congenic strain, harboring a 0.3-Mbp fragment of the chr1 QTL, has developed stroke earlier than SHRSP. Here, we investigated pathological basis of this congenic strain and attempted to identify gene(s) related to early stroke occurrence.

Methods:
SHRSP and a SHRSP-based congenic strain SHRSP.WKY-(
D1Smu13
)/Izm, abbreviated as SPwch1.71 were used in this study. The rats were housed 12-hour light phase-controlled environment with freely accessible SP diet (Funabashi Farm) and drinking water until at least one suggestive sign of stroke (diminished motor activity, paralytic gait, and so on) was found. MRI by MRmini SA 1508 (DS Pharma Biomedical) was employed to confirm stroke occurrence. BP measurement was performed by the tail-cuff method. Cerebral cortex, brainstem, kidney, adrenal gland and heart obtained from 14 weeks old rats (n=5) were snap-frozen for RNA extraction. Quantitative RT-PCR (qRT-PCR) was performed about 15 of candidate genes located on the chr1 QTL covered by SPwch1.71. Nonsynonymous SNPs were scanned based on the whole-genome sequence of SHRSP and WKY.

Results:
SPwch1.71 showed significantly high stroke susceptibility compared with SHRSP stroke-free rate until 22 weeks old; SPwch1.71: 0% (0/7), SHRSP: 62.5% (5/8),
p
<0.001. Systolic BP at 12 weeks old of SPwch1.71 was significantly higher than that of SHRSP (222±4 vs. 206±5,
p
<0.01, n=5 for each). qRT-PCR identified genes showing modest (<1.5-fold change), but statistically significant different (
p
<0.05), in the expression in each tissue examined. Nonsynonymous variations were found in
Pde2a
,
Inppl1
and
Folr1
. Pathological significance of expression/sequence variations in the genes remain unclear.
Abstract P479: Stability of Ambulatory Blood Pressure Patterns Over Time in Children and Adolescents
Authors: Hanevold Coral D , Miyashita Yosuke , Flynn Joseph T
Published: July 2021

Little is known about the stability of ambulatory blood pressure (ABP) patterns in children and adolescents undergoing evaluation for high blood pressure (HBP). It is possible that children with initially normal ABP may progress to hypertension (HT) or pre-hypertension (PHT), or that those with initial PHT or HT may be normal on repeat. Our objective was to assess stability of ABP patterns over time in children with HBP.

We analyzed changes in ABP classification in patients undergoing a minimum of 2 ABP monitoring (ABPM) studies at least 0.5 years (yrs) apart. Exclusion criteria included known secondary HT, therapy with antihypertensive medication and inadequate recordings. ABPM were interpreted according to the 2014 AHA guidelines using BP thresholds of 95
th
% for sex and height for children ≤17 yrs. For those
>
18 yrs awake and sleep thresholds were 140/85 and 120/70, respectively. Dipping was considered normal if
>
10%, blunted if <10% and reversed if < 0%. For those with > 2 ABPM the difference between the 1
st
and last were analyzed.

Two hundred ABPM on 100 patients (76 males; median age 14.6 yrs at 1st ABPM) were analyzed. Median interval between ABPM was 1.5 yrs. ABP classification was stable in 53% (53/100). As shown in the table 50% (9/18) of those with normal ABPM showed progression to PHT or HT on follow up. PreHT progressed in 31% (8/26) and improved in 38% (10/26). HT improved in 43% (20/46). Dipping designation was stable in 70% (70/100); but blunted dipping normalized in 48% (10/21).

In our population ABP patterns were not stable over time, supporting the need for follow up studies even with normal initial ABPM. If confirmed in larger studies, these findings support greater use of repeat ABPM.
Abstract P485: Use of a Noninvasive, Closed-loop, Allostatic, Neurotechnology Reduced Blood Pressure and Improved Heart Rate Variability in a Pre-hypertensive Cohort
Authors: Shaltout Hossam A , Tegeler Catherine L , Tegeler Charles H
Published: July 2021

Background:
Prehypertension increases risk for hypertension and cardiovascular disease, but effective interventions have not been defined. Disturbed central control of cardiovascular regulation due to trauma, stress, anxiety or other causes can lead to prehypertension and impaired heart rate variability (HRV). High-resolution, relational, resonance-based, electroencephalic mirroring (HIRREM®) is a noninvasive, closed-loop, allostatic, acoustic stimulation neurotechnology using software algorithms to identify specific brain frequencies, and translate them in real time into audible tones, to support self-optimization of brain rhythms.

Objective:
To evaluate the benefits of this nontraditional therapy on BP and autonomic function, subjects with untreated systolic BP of 120 to 139 or diastolic BP of 80 to 90 mmHg at baseline, who had enrolled in an IRB-approved open label feasibility study evaluating HIRREM for diverse neuropsychological disorders.

Results:
66 participants (40 female), mean (SD) age 43.3 (16.5), received 16 (5.7) HIRREM sessions over 22.4 (19.2) days, (9.5 (4.2) days with sessions). Data were collected before, and 14.4 (16.6) days after HIRREM completion. Use of HIRREM was associated with significantly reduced systolic (from 127.5 (8.0) to 122.9 (14.0) mmHg, p=0.011), and diastolic (from 82.0 (8.0) to 78.0 (9.0) mmHg, p= 0.014) arterial pressure, with no change in heart rate. HRV measured as SDNN increased (from 42.0 (17.0) to 50.0 (28.0) ms, p=0.002). Baroreflex sensitivity measured by sequence method improved (from 13.2 (8.0) to 17.2 (12.0) ms/mmHg, p=0.0001), with a trend for reduced sympathovagal tone measured by LF/HF ratio (from 2.5 (2.2) to 2.0 (1.8), p=0.068). There were no adverse events.

Conclusion:
These data provide the first report of significant cardiovascular benefits of a closed-loop allostatic therapy for prehypertension. Blood pressure reduction and HRV improvement may prevent progression to more serious cardiovascular symptoms and diseases. Further studies are indicated to investigate the mechanism of the benefits associated with this promising intervention.
Abstract P490: Dream-Global Study
Authors: Tobe Sheldon W
Published: July 2021

Introduction:
The DreamGlobal study incorporates mobile health innovations in technology with SMS text messaging and task shifting of blood pressure measurement.

Methods:
In this pragmatic RCT study participants with uncontrolled hypertension on or off of medication, received culturally competent text messages over a one year period. All participants lived on First Nations reserves in remote and rural parts of Canada. Initial blood pressure screening was with an automated oscillometric device. Community Health Workers and nurses were trained to measure blood pressure with an approved oscillometric device which was Bluetooth enabled. Once participants were registered online, they received their blood pressure results on their own mobile phone as a text message and guidelines based text messages. Their primary health care provider also received the results as a fax. They were randomized to receive health behaviour change messages alone or active messages including a recommendation to see their health care provider if their blood pressure was above target. The baseline blood pressure was the mean of all readings in the first two months and the final blood pressure, the mean of the last two months. The main outcomes were change in blood pressure between the two groups (active or passive text messages) and the proportion achieving control.

Results:
One hundred twenty-seven subjects had hypertension. Blood pressure in the first two months of measurement was 143/85 mmHg over all. The blood pressure in the final two months of the study will be reported as will the change in blood pressure in both the active and passive text message groups and the proportion achieving blood pressure control in each group. Health literacy regarding the guideline’s based text messages was measured to determine if there was a difference between scores for the passive messages (all participants) and the active messages (active group only).

Discussion:
Achieving guidelines based management of hypertension is more challenging in remote, resource poor environments. Treatment focused SMS messages may improve hypertension management and allow the provision of guidelines based care even in rural and remote areas with health care resource challenges.
Abstract P491: Characterizing Agreement in Level of Inter-extremity Blood-Pressure Readings of Adults in the Emergency Department (CALIBRATE Study)
Authors: Bhutta Zain A , Qureshi Isma , Shujauddin Mohammad , Thomas Sarah , Masood Maarij , Dsouza Landric B , Iqbal Navid , Irfan Furqan B , Pathan Sameer A , Thomas Stephen H
Published: July 2021

Introduction:
Inter-arm blood-pressure difference (IBPD) has been studied previously in multiple settings, but few reports are available from the Emergency department (ED) setting, where BP varies significantly due to acute medical conditions or stress from various factors. CALIBRATE aims to study the inter-arm blood pressure differences in the patients presenting to the ED in Qatar and to assess the IBPD distribution in this population.

Methods:
In sitting position, two consecutive BP measurements were obtained from the right and left arm for each participant using calibrated automated machines and appropriate cuff sizes. Considering the demographic mix of the population presenting to the ED, a 1:1 of male to female and 2:1 for GCC (Gulf Cooperation Council) to non-GCC recruitment strategy was predefined. The data were recorded using predefined data fields including patient demographics, past medical, social and family history. The continuous variables were reported as mean (SD) or median (IQR) based on the distribution of data. The data was analyzed using Stata MP 14.0 (College Station, Texas).

Results:
A total of 1800 patients were prospectively recruited from the ED. The mean age was 34 (10) years. The absolute systolic blood pressure (ΔSBP) difference between the right and left arm was same for the first (ΔSBP1) and the second reading (ΔSBP2), as 6 mmHg (3-10). The absolute average of ΔSBP1 and ΔSBP2

was 7 mmHg (4-10). The difference in SBP of less than 20 mmHg for IBP was seen in 95
th
percentile of the population with single reading, whereas, with the average of two individual readings it was observed in 97
th
percentile. No meaningful association could be detected between the significant IBPD

and the study variables such as age, demographics, regions of interest and risk factors. Although, patients with diagnosed hypertension met the pre-defined criterion for significance, this difference was not clinically significant. There was no significant difference between IBPD noted for the Asia-pacific or Arab population.

Conclusion:
In population presenting to the ED, the IBPD of at least 20 mmHg reached at 95
th
percentile validating the known significant difference. The utility of SBP difference can be improved further by taking the average of two individual readings.
Abstract P507: Atherosclerosis Progression in Chronic Kidney Disease
Authors: Perez Hernan A , Aballay Laura , Armando Luis J , Spence J. David , Garcia Nestor H
Published: July 2021

Cardiovascular risk is very high in CRF, but the underlying mechanisms are not well understood. Traditional cardiovascular risk factors (RF) do not explain the increased risk, and observational studies have observed paradoxical or absent associations between classical RF and mortality in ESRD. CRF studies found that statin therapy does not reduce CV events; these may be the results of “resistant atherosclerosis” observed in these patients. We investigated if carotid total plaque area (TPA) is increased at progressively lower creatinine clearance and whether or not TPA progression is increased in CRF patients not on dialysis.

Methods:
The Blossom DMO Argentina ethics committee approved the study and informed consent was obtained from each participant. We performed a cohort study in 201 patients with Normal Renal Function (NRF), Stage 2 and 3 CRF. Clinical, laboratory tests and TPA were determined at time 0 and after 1 year. TPA was measured using carotid ultrasonography. Renal function (eGFR) was determined by the MDRD equation. The Study population was divided into quartiles of eGFR.

Results:
1
st
Quartile, (51±1yo, eGFR 89±2 ml/min) had a blood pressure (BP) of 136±2/81±1 mmHg, BMI 31±1, Total Chol (tChol) 196±6 mg/dl, HbA1c 6.7±0.4% and had the lowest Chol 192±5 mg/dl, HbA1c 6.2±0.1% and TPA 47±6mm
2
; 3
rd
Quartile, (59±1yo, eGFR 63±1 ml/min) BP 133±2/82±1, tChol 192±5 mg/dl, HbA1c 6.2±0.1% and TPA 47±6mm
2
; 4
th
Quartile (60±2yo, eGFR 52±1 ml/min) BP 140±3/84±1, tChol 209±5 mg/dl, HbA1c 6.2±0.1% and TPA 76±11mm
2
. After one year, the 4
th
Quartile had the most progression of TPA (
p
< 0.005); it was not influenced by age, hypertension, smoking, dyslipidemia or diabetic status.

Conclusions:
In CRF, TPA increases as renal function decreases; its progression is not associated with traditional risk factors. Other mechanisms are responsible for the observed excess of cardiovascular disease in CKD. Determination of TPA should be used to measure effects of antiatherosclerotic therapy to decrease the enormous cardiovascular event rate observed in this population.
Abstract P527: Angiotensin-(1-7) Attenuates Doxorubicin-Induced Aortic Dysfunction in Male and Female Rats by Distinct Mechanisms
Authors: Rahimi Omeed , Kirby Jay L , Varagic Jasmina , Tallant E. Ann , Gallagher Patricia E
Published: July 2021

Doxorubicin (Dox), a commonly used and effective chemotherapeutic agent, often produces cumulative dose-dependent cardiovascular toxicity, resulting in long-term hypertrophy and fibrosis which can lead to heart failure. Adjunct therapies are thus needed to reduce Dox-induced cardiovascular toxicity and enhance long-term quality-of-life in cancer patients, especially in pediatric patients. Angiotensin-(1-7) [Ang-(1-7)] is an endogenous peptide hormone of the renin-angiotensin system that improves cardiac and vascular function by reducing hypertrophy and fibrosis in various animal models. In this study, juvenile Sprague-Dawley rats (male and female, n = 8-10) were administered Dox (cumulative dose of 22 mg/kg) for 6 week, in the presence and absence of Ang-(1-7) [24 μg/kg/h]. Aortic function was measured using a Vevo 2100 small animal ultrasound system. In both males and females, Dox administration increased pulse wave velocity (PWV), a measure of arterial stiffness, and co-treatment with Ang-(1-7) attenuated the Dox-induced increase (Males - 5.6 ± 0.5, Sham; 9.7 ± 1.4, Dox; 7.8 ± 0.6 m/s, Dox/Ang-(1-7), p < 0.01; Females - 5.1 ± 0.5, Sham; 14.3 ± 1.5, Dox; 7.7 ± 1.2 m/s, Dox/Ang-(1-7), p < 0.001); Ang-(1-7) alone had no effect. Dox increased aortic thickness and decreased aortic diameter at systole in males only, which was attenuated by Ang-(1-7) (aortic thickness - 0.28 ± 0.01, Sham; 0.33 ± 0.01, Dox; 0.28 ± 0.01 mm, Dox/Ang-(1-7), p < 0.01; aortic diameter - 2.8 ± 0.6, Sham; 2.3 ± 0.1, Dox; 2.5 ± 0.1 mm, Dox/Ang-(1-7); p < 0.01). No change in aortic thickness or diameter was observed following treatment with Ang-(1-7) alone. Conversely, Dox increased fibrosis in female aorta only, measured by immunohistochemistry with Picrosirius red, which was attenuated by Ang-(1-7) (5.4 ± 0.3, Sham; 7.2 ± 0.6, Ang-(1-7); 12.8 ± 2.0, Dox; 8.0 ± 1.0%, Dox/Ang-(1-7); p < 0.001). These results demonstrate that Dox causes aortic dysfunction in both males and females, albeit through different mechanisms—an increase in aortic hypertrophy in males and aortic fibrosis in females. Ang-(1-7) attenuated both the hypertrophy and fibrosis, suggesting that treatment with the heptapeptide hormone may serve as an effective adjuvant to improve Dox-induced aortic dysfunction.
Abstract P533: Distinct Molecular Compositions of Circulating Microparticles in Hypertension and Diabetes
Authors: Turner Maddison , Thibodeau Jean-Francois , Holterman Chet , Kennedy Christopher , Burger Dylan E
Published: July 2021

Background:
Microparticles (MPs) are emerging as novel markers and mediators of vascular pathology. The purpose of this study was to examine the effect of hypertension and diabetes on the molecular composition of circulating MPs.

Methods:
We studied circulating MPs isolated from transgenic mice expressing active human renin in the liver (TtRhRen, a model of hypertension), OVE26 type 1 diabetic mice, and their wild-type (WT) littermates. At 20 weeks of age mice were sacrificed and blood samples were obtained by cardiac puncture. MPs were subsequently isolated from platelet-free plasma via differential centrifugation and protein content was assessed via mass spectrometry (MS).

Results:
TtRhRen mice exhibited increased blood pressure compared with OVE26 mice or their WT littermates. (144.2±7.6 vs 123.5±4.9 [OVE26] vs. 114.6±5.7 mmHg [WT], p<0.05). MS identified 300 independent proteins with at least 2 peptides per protein. Of these, 167 were found in all groups studied, 27 were exclusive to WT mice, 4 were exclusive to TtRhRen mice and 46 were exclusive to OVE26 mice. In addition, 16 proteins were found in MPs from WT and OVE26 mice but absent from TtRhRen mice while 40 were found in MPs from TtRhRen and OVE26 mice but absent in MPs from WT mice. Interestingly, proteins exclusive to MPs derived from hypertensive TtRhRen mice included two proteins previously implicated in the pathogenesis of hypertension: insulin-like growth factor-binding protein 5 and haptoglobin.

Conclusions:
Taken together our results suggest that circulating MPs display a distinct molecular composition, reflective of pathogenic state. Further examination of these changes may lead to the identification of novel biomarkers of vascular injury in hypertension and diabetes.
Abstract P539: Cardiac and Renal Involvement in Malignant-accelerated Hypertension: Implications on Long-term Survival
Authors: Bortolotto Luiz , De Lima Jose J , Giorgi Dante
Published: July 2021

Background:
Malignant hypertension is a severe complication of arterial hypertension where end-organ damage is severe and occurs in a short period of time. Data about cardiac and renal involvement and implications on long-term survival of patients with malignant hypertension are scarce.

Methods:
We performed a single-centre retrospective analysis of 176 patients with malignant hypertension, diagnosed in the period 1984-2007.

Results:
Incidence of malignant hypertension decreased along the different periods of the study, but cardiac and renal damage at presentation were common and severe. In 94.6% we observed left ventricle hypertrophy on echocardiogram, in 83.2% the glomerular filtration rate was below 60 ml/min/1.73m2 and 11% of patients required immediate dialysis. The survival rate was 95% at 6 months, 90% at 12 months, 81% at 36 months, and 67% at 5-year follow-up. In a Cox-regression analysis, the independent predictors of all-cause mortality were age, SBP at discharge, septum wall thickness values and GFR at admission.

Conclusion:
Malignant hypertension remains a severe complication of arterial hypertension with a high mortality rate at 5-year, that was independently associated to the severe cardiac and renal involvement present in most patients.
Abstract 1: Opposing Roles for Endoglin and Soluble Endoglin in Cardiac Remodeling and Heart Failure
Authors: Kapur Navin K , Wilson Szuhuei , Yunis Adil A , Baker Corey , Aronovitz Mark J , Lu Qing , Karumanchi Ananth , Letarte Michelle , Kass David A , Mendelsohn Michael E , Karas Richard H
Published: July 2021

Transforming growth factor beta-1 (TGFb1) promotes cardiac fibrosis. The transmembrane co-receptor Endoglin (Eng; CD105) facilitates TGFb1 signaling via SMAD effector proteins. In contrast, a circulating form of soluble endoglin (sEng) inhibits TGFb1 signaling in vascular endothelium. We recently reported that increased sEng levels in human serum correlate with clinical indices of heart failure severity. Therefore, we tested the hypothesis that Eng and sEng mediate opposing effects on cardiac fibrosis in heart failure. In male, wild-type mice (WT), Eng expression increased in the left ventricle (LV) after 2, 4, and 10 weeks of thoracic aortic constriction (TAC) accompanied by progressive LV fibrosis and hypertrophy. In contrast to WT mice, Eng haploinsufficient (Eng
+/−
) mice had preserved LV function (FS%: 78±4 vs 22±16, Eng
+/−
vs WT, p<0.01) and improved survival [88%(7/8) vs 50%(4/8), Eng
+/−
vs WT, p<0.001) after 10 weeks of TAC. Reduced LV fibrosis was observed in Eng
+/−
mice, while LV mass, cardiomyocyte hypertrophy, and calcineurin, SerCA, and bMHC expression were comparable to WT after TAC. Capillary density was significantly higher in Eng
+/−
mice after TAC compared to WT. LV SMAD phosphorylation (pSMAD) after TAC was studied and a preferential increase was observed in pSMAD1/5/8 expression in Eng
+/−
mice as compared to WT mice, in which cardiac pSmad2/3 expression was increased. The dependence of TGFb1 induced collagen synthesis on Eng expression was tested in vitro using human cardiac fibroblasts (hCF). Neutralizing antibodies and siRNA against Eng each attenuated TGFb1 induced collagen synthesis. In contrast, conditioned media from cells transfected with an adenovirus over-expressing sEng attenuated pSMAD2/3 expression and TGFb1 induced collagen synthesis in hCF. Treatment of hCF with recombinant sEng blocked TGFb1 induced collagen synthesis in a dose-dependent manner, confirming an inhibitory role of sEng. These results indicate that Eng and sEng mediate opposite effects on TGFb1 induced collagen synthesis. Reduced Eng expression uncouples cardiac fibrosis from cardiomyocyte hypertrophy, promotes SMAD1/5/8-signaling, and enhances angiogenesis. Eng may represent a novel therapeutic target to improve survival in heart failure.
Abstract P032: Nanoparticle Mediated Nox2-siRNA Therapy for Preventing Cardiac Dysfunction Following Myocardial Infarction
Authors: Somasuntharam Inthirai , Murthy Niren , Davis Michael
Published: July 2021

Myocardial infarction (MI) is the most common cause of heart failure in the developed world. Following MI, reactive oxygen species (ROS) play a key role in the pathogenesis of cardiac remodeling leading to impaired ventricular function. Oxidative stress at high levels lead to many of the injury associated changes: proinflammatory cytokine release, myocyte apoptosis, cardiac fibrosis and hypertrophy. Nicotinamide adenine denucleotide phosphate (NADPH) oxidase with Nox2 as the catalytic subunit, is a major source for cardiac ROS production. After MI, Nox2 expression is significantly increased in the infarcted myocardium. Moreover, mice lacking the Nox2 gene are protected from ischemic injury. We used polyketals, a new class of acid-degradable polymers, as delivery vehicles for Nox2-siRNA to the post-MI environment. When engaged by macrophages, present in high quantities during MI, these particles have been shown to be taken up by macrophages and contents released within cells in active form. Nox2-siRNA was ion-paired to the cationic lipid DOTAP, and spherical particles averaging 500nm diameter were made by a single emulsion procedure with polyketal PK3 using PVA as surfactant. While a commercially available transfection reagent yielded 7% uptake as measured by flow cytometery, 81% of macrophages were positive for fluorescently-labeled siRNA in PK3-siNOX2 treated cells. These data were confirmed with confocal microscopy. Macrophages treated with PK3-siNOX2 demonstrated a significant 43% knockdown in Nox2 gene expression at 24 hours, while no reduction was seen with scrambled siRNA or empty particles. Functional activity was assessed by a fluorescent dihyroethidium dye based HPLC quantification after phorbol 12-myristate 13-acetate stimulation following 72 hours of particle treatment to measure superoxide production. PK3-siNox2 treated cells exhibited a 41% reduction in activity, with no significant changes seen with scrambled siRNA or empty particle treatment. Currently, the therapeutic potential of the Nox2-siRNA particles is being evaluated in a mouse model of ischemia-reperfusion. Successful completion of these studies could lead to a novel treatment for post-infarction injury.
Abstract P046: Hsp20 Promotes Cardiac Autophagy via Interaction with Beclin-1
Authors: Zhu Hongyan , Wang Xiaohong , Zhang Xiaowei , Kranias Evangelia G , Liang Qiangrong , Fan Guo-Chang
Published: July 2021

Hsp20 has been shown to prevent stress-triggered cardiac injury. However, it remains obscure whether Hsp20-elicited cardioprotection is associated with the activation of autophagy. We first assessed cardiac autophagy in a transgenic (TG) mouse model with 10-fold overexpression of Hsp20 by immunoblotting. Levels of two autophagy markers (LC3II and Beclin1) were increased by 1.6- and 2.2-fold, respectively, in Hsp20-hearts, compared to WTs. To examine whether Hsp20 activates autophagic flux in the heart, mice were
i.p.
injected with chloroquine (CQ, 50mg/kg), an inhibitor for autophagosome-lysosome fusion, for 4h. Cadaverine dye-binding analysis showed that autophagy levels were increased by 1.5-fold in CQ-treated Hsp20-hearts compared to saline controls. To examine whether increased autophagy levels contribute to Hsp20-induced cardioprotection, 3-methyladenine (3-MA, an inhibitor for autophgy) was
i.p
. injected into TG mice (30mg/kg). One hour later, hearts were subjected to global no-flow ischemia/reperfusion (I/R: 45min/1h), and showed that contractile function (+dP/dt) was recovered by 93α 5 % in saline-treated Hsp20-hearts, comparable to 74 α 6 % recovery in 3-MA-treated TGs (n=6, p<0.05), indicating that the activation of autophagy by Hsp20 is beneficial for hearts upon I/R. To exclude
in vivo
compensatory effects, Hsp20 was delivered into isolated adult cardiomyocytes by adenoviral vector. Western-blotting analysis indicated that acute expression of Hsp20 increased the levels of LC3II and Beclin1 by ∼2-fold, compared to GFP control. Accordingly, overexpression of Hsp20 augmented myocyte survival upon addition of H
2
O
2
, as related to GFP-control. Furthermore, co-infection with AdBeclin1-siRNA attenuated the protective effects of Hsp20 in myocytes upon H
2
O
2
stress. Excitingly, Hsp20 was found to localize at the LC3-labeled autophagosome. Indeed, co-immunoprecipitation results showed an interaction of Hsp20 with Beclin-1 in the heart. Moreover, a competitive ELISA assay revealed that Hsp20 dose-dependently suppressed the interaction of Beclin1/Bcl-2, a known complex negatively regulating autophagy. In summary, Hsp20 activates cardiac autophagic flux via interaction with Beclin1, thereby confering cadioprotection.
Abstract P056: RalGDS-Dependent Cardiomyocyte Autophagy in Load-Induced Ventricular Hypertrophy
Authors: Rifki Oktay F , Bodemann Brian O , White Michael A , Hill Joseph A
Published: July 2021

Background:
Recent work has demonstrated that autophagy, a phylogenetically conserved, lysosome-mediated pathway of protein degradation, is a key participant in pathological cardiac remodeling. One common feature of cell growth and autophagy is membrane biogenesis and processing. The exocyst, an octomeric protein complex involved in vesicle trafficking, is implicated in numerous cellular processes, yet its role in cardiomyocyte plasticity is unknown. Here, we set out to explore the role of small G protein-dependent membrane trafficking in stress-induced cardiomyocyte remodeling and autophagy.

Methods and Results:
Hearts from mice lacking RalGDS (
Ralgds
-/-
), a guanine exchange factor (GEF) for the Ral family of small GTPases, were similar to wild-type (WT) littermates in terms of ventricular structure, contractile performance, and gene expression. However,
Ralgds
-/-
hearts manifested a blunted growth response (p<0.05) to TAC-mediated pressure-overload stress as determined by heart weight to body weight ratios (HW/BW; WT, sham: 5.00 ± 0.21 mg/g, n=9; TAC: 6.93 ± 0.29, n=13;
Ralgds
-/-
sham: 4.87 ± 0.19, n=6, TAC: 5.86 ± 0.10, n=6). Ventricular chamber size and contractile performance were unchanged in response to TAC in both genotypes. Interestingly, TAC-induced activation of the fetal gene program was similar in both genotypes despite the relative lack of hypertrophic growth in mutant hearts.
Ralgds
-/-
mice also exhibited diminished load-induced cardiomyocyte autophagy. Consistent with the TAC findings,
Ralgds
-/-
mice manifested a blunted autophagic response to 24-hour fasting, suggesting a generalized defect in autophagy. To explore underlying mechanisms, we tested in cultured neonatal cardiomyocytes two isoforms of Ral that are downstream of RalGDS (RalA, RalB) and whose actions are mediated by the exocyst. In these experiments, mTOR inhibition was maintained in response to starvation and rapamycin despite RalA or RalB knockdown; however, autophagy was diminished only in NRCM's with RalB knockdown, implicating RalB as required for cardiomyocyte autophagy.

Conclusions:
Together, these data implicate RalGDS-mediated induction of autophagy as a critical feature of load-induced cardiac hypertrophy.
Abstract P066: Imaging Autophagy in Living Mice
Authors: Gottlieb Roberta A , Sayen M R , Huang Chengqun , Ramil Jennifer , Ito Bruce , Mentzer Robert M
Published: July 2021

Autophagy is a homeostatic response to cellular stress. It has been shown to be potently upregulated in the heart in response to a variety of interventions. However, to date, it has not been possible to monitor autophagy without sacrificing the animal. Here we report the use of the Caliper Life Sciences Spectrum In Vivo Imaging System (IVIS) to image autophagy in homozygous transgenic mice expressing mCherryLC3 under control of the alpha myosin heavy chain promoter. Autophagy was stimulated by the administration of rapamycin (2mg/kg), and autophagosomal flux was blocked by administration of chloroquine (10mg/kg) ip. Mice were imaged at baseline and four hours later, using a protocol of 3 acquisitions of 15 seconds each. Total flux was 3.19±0.72 before drug administration and 3.93+1.10 after 4 hr, p<;0.01, n=14. These results show for the first time imaging of autophagy in hearts of live mice.
Abstract P080: Bestrophin: A Potential Mediator of Cardiac Calcium Handling
Authors: Britton Fiona C , John Steeven , O'Driscoll Kate E , Baring Martha
Published: July 2021

Bestrophins are a family of transmembrane proteins expressed in heart. We reported that Bestrophin 1 and 3 cloned from mouse heart function as calcium-activated chloride channels. We hypothesized that bestrophin channels may function in a macromolecule complex similar to other cardiac ion channels. To identify proteins that bind Bestrophin 3 channel, we screened a mouse heart cDNA library using 4 different cytosolic cDNA fragments as bait. Yeast two-hybrid assays were performed with the GAL4 system. Using bait consisting of C-terminus amino acid residues 386 to 669 of the Best3 channel (Best3-C2), we obtained positive interactions with the histidine-rich calcium binding protein (hrc). Hrc is a component of the SR complex involved in calcium handling in the heart. Eleven independent clones represented a fragment encoding the cysteine rich domain of hrc previously shown to interact with triadin. To further map the interaction sites between Best3-C2 and hrc, we subdivided this region into three fragments and tested these baits for interaction with hrc. Two Best3 fragments (residues 541–600 and residues 601–669) displayed strong interaction with hrc, while Best3 bait (residues 386–540) failed to interact. This region of Best3 contains a KEKE protein binding motif. In a similar manner C-terminal Best 1 fragments were confirmed to also interact with hrc. In this study we report that Bestrophin channels interact with hrc, a cardiac calcium handling protein. Calcium-activated chloride channels play a critical role in excitation-contraction coupling in the SR by balancing charge movement during calcium release and reuptake. Recent evidence confirms Bestrophin 1 is located in the ER membrane in association with Stim1 and facilitates calcium cycling. Future in vivo investigations will examine the role of bestrophins as potential mediators of calcium handling in heart.
Abstract P082: Cardiac Calcium Handling and Protein Phosphatase 2A Activity Are Differentially Regulated by JNK1 and JNK2 MAP Kinases
Authors: Golden Honey B , Watson Linley E , Foster Donald M , Dostal David E
Published: July 2021

We previously identified the JNK-B56α-PP2A signaling axis as a major target in anthrax lethal toxin (LT)-induced cardiac dysfunction. Thus, we further tested whether LT-mediated loss in cardiac function is a consequence of dysregulated calcium handling resulting from JNK inactivation. To biochemically recapitulate the signaling effects of LT, we infected NRVM with HA-B56α adenovirus and determined PP2A activity as well as Ca
2+
i
measurements. Over-expression of HA-B56α in NRVM did not induce a significant increase in cellular PP2A activity, however, it did induce a significant increase (p<0.01) in Ca
2+
i
compared to virus control. Furthermore, PLB, PP2Ac and Akt protein co-immunoprecipitated with HA-B56α, and immunostaining revealed colocalization of B56α with PLB at the SR. Since B56α over-expression alone was not sufficient to induce PP2A activity or PP2A-mediated Ca
2+
i
dysregulation, we hypothesized that JNK may serve as a functional regulator of Ca
2+
i
handling through PP2A activation as well as B56α protein levels. Adenoviral-mediated over-expression of MEK7 in NRVM resulted in a significant reduction in LT-mediated Ca
2+
i
dysregulation compared to virus control (p<0.01). To further determine whether the protection of MEK7 is mediated by JNK1 or JNK2, gain-of-function/loss-of-function experiments were performed utilizing adenoviral constructs for CA-MEK7, DN-JNK1 and DN-JNK2. Results confirmed that the protective effect of active MEK7 over-expression on Ca
2+
i
was significantly lost with knock-down of JNK1 compared to JNK2 (p<0.05), suggesting that JNK1 plays a more substantial role in regulating PP2A activity than JNK2. Furthermore, the selective knock-down of JNK1 also increased Ca
2+
i
levels (p<0.001) compared to MEK7 during LT treatment, which reveals the importance of JNK1 in PP2A-mediated Ca
2+
i
dysregulation. Interestingly, immunoblotting of PLB did not reveal a JNK-dependent difference in PLB phosphorylation at Ser
16
, however, loss of JNK2 almost completely inhibited p-PLB-Thr
17
. Thus, our results suggest that JNK1 and JNK2 may differentially regulate Ca
2+
i
through PP2A activation and PLB-Thr
17
phosphorylation, respectively.
Abstract P088: The Rate of Inorganic Phosphate Release in Contracting Rat Trabeculae Is Independent of Calcium After Stretch
Authors: Mansfield Catherine , West Timothy , Ferenczi Mike
Published: July 2021

We aim to reveal the molecular mechanism underlying the force response to stretch in the heart by examining the dependence of the rate of inorganic phosphate (P
i
) release in response to stretch on the degree of calcium activation. Permeabilized trabeculae of rat heart are activated by photolytic release of ATP, at 20°C, in the presence of saturating (32µM), or half-saturating (1µM), calcium concentrations, at an initial sarcomere length of 1.9 or 2.1 µm. For trabeculae with an initial sarcomere length of 1.9 µm, the trabeculae are stretched to 2.1 µm during the isometric plateau, held at a constant length and returned to their original length. The rate of P
i
release is determined by the fluorescence change associated with phosphate binding to a fluorescently-labelled phosphate binding protein diffused into the preparation. At an initial sarcomere length of 1.9µm, the isometric force at full activation was 51 kN.m
−2
compared to 26kN.m
−2
at half-maximal activation. During the isometric phase, P
i
release is at a steady rate of 8.2 and 4.8 s
−1
(assuming a myosin head concentration of 120µM) at 32 and 1µM calcium, respectively. During stretch, the rate of P
i
release decreases markedly at both activation levels, to1.0 and 1.4s
−1
respectively. Activation from an initial sarcomere length of 2.1µm produced an isometric force of 65.5 kN.m
−2
with a steady P
i
release rate of 8.1 s
−1
at maximal activation, and an isometric force of 49.2 kN.m
−2
with a steady P
i
release rate of 4.8 s
−1
at half-maximal activation. The rates of P
i
release during stretch and after the end of the stretch at low activation are equal to that at high activation indicating stretch-induced activation which occurs for stretches applied prior to activation, and during activation. The results demonstrate that stretch has a dramatic effect causing an immediate reduction in the rate of P
i
release. Stretch contributes to activation of the trabecula's actomyosin ATPase in a calcium-independent manner, although the effect is more marked at low activation levels. Thus a direct effect of stretch on thin filament activation contributes to the Frank-Starling law.