bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract P176: Familial Autonomic Ganglionopathy Caused by Rare CHRNA3 Genetic Variants. Novel Genetic Cause of Neurogenic Orthostatic Hypotension
Authors: Shibao Cyndya A , Phillips John , Cogan Joy D , Newman John H , Hamid Rizwan , Meiler Jens , Capra John A , Sheehan Jonathan , Yan Yaping , Vetrini Francesco , Black Bonnie , Joos Karen , Robertson David , Biaggioni Italo
Published: September 2019

Patients with autonomic failure developed severe neurogenic orthostatic hypotension (nOH). There are only two reported genetic conditions that cause nOH, dopamine β-hydroxylase deficiency, and CYB561 deficiency; both affect norepinephrine synthesis resulting in an isolated sympathetic failure.

Objective:
To determine molecular basis of a new Mendelian cause of familial nOH with impaired cardiovagal function, and miosis associated with sympathetic (SNS) and parasympathetic (PNS) failure.

Method and Results:
The proband developed disabling nOH with poor compensatory increase in heart rate at 39 yrs of age. Plasma norepinephrine levels were abnormally low. His sister had onset of nOH in her late teens. Their parents and brother were healthy. Whole exome sequencing of DNAs from the proband and his affected sister showed that both were compound heterozygotes for c.907_908delCT (p.L303Dfs*115) /c.688G>A (p.D230N) variants in the acetylcholine receptor, neuronal nicotinic, alpha 3 subunit (CHRNA3) gene. CHRNA3 is a subunit of nicotinic acetylcholine receptors (nAChRs) that regulates blood pressure through modulation of synaptic transmission in the autonomic ganglia. The autonomic ganglia regulates both SNS and PNS activity. The frameshift (fs) and missense CHRNA3 variants were inherited from the sibs’ mother and father, respectively, and their non-affected brother was a nl/p.D230N heterozygote. The fs variant is obviously pathogenic and the p.D230N variant is predicted to be damaging (SIFT)/probably damaging (PolyPhen2). Furthermore, we predict from structural modeling that the p.D230N variant lies on the interface between CHRNA3 and other nAChR subunits and that it would destabilize the nAChR pentameric complex. In humans, the presence of acquired nAChR autoantibodies against the alpha 3 subunit is associated with nOH and non-reactive pupils.

Conclusions:
We report sibs affected with NOH and miosis who are compound heterozygotes forCHRNA3 variants that co-segregate with and have predicted effects on nAChR structure that suggest they likely cause this family’s SNS and PNS failure.
Abstract P180: Deficiency of Sirtuin 3 Sensitizes Angiotensin II-Induced Cardiac Pericyte-Fibroblast Transition and Diastolic Dysfunction via Iron Overloading
Authors: Su Han , zeng Heng , Worsham Rebecca A , Chen Jian Xiong X
Published: September 2019

Hypertension is the key factor for the development of cardiac fibrosis and diastolic dysfunction. Our previous study showed that knockout of SIRT3 resulted in diastolic dysfunction in mice. In present study, we aim to explore the role of SIRT3 in pericyte recruitment and cardiac fibrosis in response to hypertension. NG2
+
tracing reporter NG2DsREDBAC mouse was crossed with SIRT3KO mice. Mice were infused with Ang-II (1000ng/kg/min) for 28 days, blood pressure, coronary flow reserve (CFR) and diastolic function (IVRT and MPI) were measured pre- and post- Ang-II treatment. Cardiac fibrosis and iron content were measured by Masson and Prussian staining. Pericytes and fibroblasts were measured by co-immunostaining. The expression of heme oxygenase-1 (HO-1), ferroportin (FPN), p47
phox
, and transforming growth factor-β1(TFG-β1) was analyzed by western blots. Infusion of Ang-II resulted in a significant increase in blood pressure compared to basal levels in WT mice (MAP 94± 2 mHg vs 130±1.7 mHg, n=7, p<0.05); whereas knockout of SIRT3 exacerbated Ang-II-induced increase in blood pressure (MAP WT 130±1.7 mHg vs SIRT3KO 145 ± 2.1 mHg, n=7, p<0.05). Furthermore, knockout of SIRT3 sensitized Ang-II-induced reduction of CFR and elevation of IVRT and MPI in mice. Using NG2 pericyte tracing reporter mice, we found that mice infusion with Ang-II resulted in a significant increase in NG2
+
pericyte recruitment in the heart. Knockout of SIRT3 enhanced Ang-II-induced NG2
+
pericyte recruitment. Knockout of SIRT3 further enhanced Ang-II-induced cardiac fibrosis as well as fibrotic markers FSP1 and a-SMA staining. To examine whether pericytes differentiate into fibroblast, NG2
+
pericytes were co-stained with FSP-1
+
. Ang-II infusion led to a significant increase in NG2
+
/FSP-1
+
; while knockout of SIRT3 further increased numbers of NG2
+
/FSP-1
+
in the heart. Mechanistically, knockout of SIRT3 increased iron staining in the heart. This was accompanied by a significant increase in the expression of HO-1, FPN, p47
phox
, ROS, and TGF-β as compared to Ang-II treatment. Our study suggests that deficiency of SIRT3 may promote Ang-II-induced cardiac fibrosis via pericyte-fibroblast transition and by Fe2
+
-ROS-TGF-β pathway.
Abstract P182: Quantifying Differences in Blood Pressure by Using Different Blood Pressure Cuff Sizes in Children
Authors: Mahajan Ruchi Gupta , Iqubal Kashif , Kern Jeffrey
Published: September 2019

Background:
The prevalence of clinical HTN in children and adolescents is 3.5%. Measuring BP with an inappropriate cuff is the most common cause of misdiagnosing hypertension. Incorrect measurement can result in unnecessary investigations, treatment, and follow-up for the mistaken diagnosis of hypertension, or no treatment for hypertension in individuals mistakenly thought to have normal BP.

Methods:
A Prospective case control study of healthy children 4-12 years of age visiting Flushing Hospital Medical Center ACC, between Jan-Nov 2018. Data collected included age, gender, ethnicity, body mass index (BMI), arm circumference, and BP values obtained with different size cuffs. Subjects were divided into 4 BMI groups, differentiated as underweight (B1, BMI < 18.5), normal weight (B2,18.5-24.9), overweight (B3,25-29.9), or obese( B4, > 30).

Result:
The study was conducted with 137 patients with average age of 7.67 ± 2.48 (Median age 7.0). The study sample included 64 (43%) females and 73 (57%) males with 91% were Hispanic. Two patients were obese and given the limited sample size were excluded.The systolic BP was found to be 5 mmHg less with the larger cuff and 5 mmHg more with the smaller cuff. A one-way ANOVA was done to compare the effect of age on changes in systolic BP when a larger cuff was used. These was no statistical significance between changes in systolic BP for the different age groups (F(8,125)=1.318,
p
=0.2407). A one-way ANOVA was done to compare the effect of BMI on changes in systolic BP when a larger cuff was used. There was no statistical significance between changes in systolic BP for the BMI groups (F(2,131)=2.349,
p
=0.09). The same was done for the smaller cuff and systolic BP. No statistically significant change was found because of age (F(8,113)=0.7423,
p=0.65
) or BMI (F(2,119)=2.916,
p
=0.05). No statistical/clinical difference was found when measuring diastolic BP.

Conclusions:
Systolic BP in children aged 4-12 years was 5 mmHg higher when measured with a
smaller cuff and
5 mmHg lower when measured with a
larger cuff.
Diastolic BP was not clinically significant for different cuff sizes. Age and BMI did not affect the differences in BP measurement.
Abstract P184: Exploring Sex Differences in Immune Cell Profiles of Male, Premenopausal Female, and Postmenopausal Female Mice to Understand Susceptibility to Immune Mediated Hypertension
Authors: Sylvester Megan A , Pollow Dennis P , Uhlorn Josh A , Romero-Aleshire Melissa J , Uhrlaub Jennifer , Nikolich-Zugich Janko , Brooks Heddwen L
Published: September 2019

T cells are required for the development of hypertension in male and postmenopausal female mice while premenopausal females are protected from T cell mediated hypertension. To better understand sex differences in immune-cell mediated hypertensive responses, we sought to determine if there were any significant differences in the immune cell profiles of premenopausal female (F), VCD-treated postmenopausal female (PMF), and male (M) mice. Spleens were collected from all mice and processed for flow cytometric analysis of T cell populations (n=8/group). Analysis of splenic T cell populations revealed no significant difference in the frequency of CD3+ or CD4+ T cells between groups (CD3+: F 33.4%, PMF 30.3%, M 30.2% of total lymphocytes, CD4+: F 64.8%, PMF 70.7%, M 67.5% of CD3+ cells). However, postmenopausal females had a significantly lower frequency of splenic CD8+ T cells compared to both males and premenopausal females (CD8+: F 27.9%, PMF 19.5%*, M 25.3% of CD3+ cells *p<0.05 vs M and F). Additionally, premenopausal females displayed significantly increased expression of the memory marker CD44 and the anti-inflammatory marker CTLA-4 on CD4+ cells compared to both males and postmenopausal females (MFI CD44: F 334.8*, PMF 269.4, M 280.4, MFI CTLA-4: F 100.7*, PMF 80.9, M 86.8 *p<0.01 vs M and PMF). Additional flow cytometric staining was performed to evaluate sex differences in splenic Antigen Presenting Cell (APC) populations (n=8/group). The frequency of CD11b+ APCs, thought to primarily represent macrophage populations, were significantly reduced in postmenopausal females compared to premenopausal females but there was no significant difference from males (CD11b +: F 14.6%, PMF 11.8%*, M 12.9% of monocytes *p<0.05 vs F). Additionally, CD11c+ dendritic cell populations were found to be significantly reduced in postmenopausal females compared to both males and premenopausal females (CD11c+: F 4.1%, PMF 2.8%*, M 3.9% of all monocytes *p<0.01 vs M and F). Taken together, these results indicate a significant difference in the baseline immune environment between male, premenopausal female and postmenopausal females which likely contribute to sex-differences in susceptibility to immune-mediated hypertension.
Abstract P186: Specific Knockout of Sox6 in Renin Expressing Cells Cause a Decrease in Renin Expression and Changes to Vascular Reactivity
Authors: Saavedra Luz , Saleem Mohammad , Xiao Liang , Ao Mingfang , Gomez Jose A
Published: September 2019

Introduction:
Sox6 is a transcription factor known to be important for specification of different cell lineages, and important during embryonic development in vertebrates. Our group recently found that Sox6 plays a new function in renin expression control. Renin is an enzyme that controls the rate limiting step in the Renin Angiotensin Aldosterone System, a key regulator of blood pressure and thus hypertension. Here we report new findings in a novel transgenic mouse in which Sox6 is specifically knocked out in Renin expressing cells (Ren1d
Cre
/Sox6
fl/fl
-Sox6 KO).

Methods:
Sox6 KO was used to determine the impact of Sox6 ablation in renin expressing cells on renin expression. Blood pressure and heart rate variability were measured by telemetry. Vascular reactivity was measured by wire myography and sympathetic nervous system renal input was determined by renal catecholamines assay.

Results:
We found that the specific Sox6 ablation in renin expressing cells produced a significant decrease in renin expression at a protein (40% fold decrease, n=7, P=0.01) and mRNA level (37 % fold decrease, n=4, P=0.01) compared to wild type mice. Our data indicates that Sox6 KO has no significant change in baseline blood pressure. However, there is a decrease in heart rate variability in Sox6 KO mice compared to wild type, indicating a dysregulation in the sympathetic and parasympathetic nervous systems (3.22±0.19 vs 1.14±0.32 LF/HF, n=10-13, P<0.05). Regarding vascular function, Sox6 KO mice have an enhanced sensitivity to vasodilators compared to wild type mice which may compensate for the decrease in renin expression (difference in response to sodium nitroprusside was found between 3 nM and 30 nM, n=3-11, P<0.05) and no difference in Acetylcholine response at any concentration. Furthermore, preliminary data indicates that there are no detectable differences in renal norepinephrine concentration between Sox6 KO and wild type mice.

Conclusion:
Our data indicate that Sox6 has a new function in renin-expressing cells that modulate blood pressure, heart rate variability, and vascular reactivity. Currently we are studying changes in the expression of vascular tone genes in endothelial and smooth muscle cells and changes in the sympathetic tone in the kidney and vascular system.
Abstract P189: Human Endothelin-1 Overexpression in Mice Changed Gene Expression in Mesenteric Arteries
Authors: Berillo Olga , Coelho Suellen C , Huo Ku-Geng , Ouerd Sofiane , Richer Chantal , Mahjoub Nada , Ferreira Nathanne S , Sinnett Daniel , Paradis Pierre , Schiffrin Ernesto L
Published: September 2019

Background:
Transgenic mice with tamoxifen-inducible endothelium-restricted human endothelin-1 overexpression (ieET-1) exhibited blood pressure (BP) elevation for 3 weeks or 3 months after induction. Vascular injury was observed only after 3-month exposure to human ET-1 overexpression. It is unknown whether 3-week or 3-month exposure to ET-1 overexpression results in gene dysregulation. We aimed to identify differentially expressed (DE) microRNAs (miRs) and genes in mesenteric arteries (MAs) of ieET-1 mice after 3-week and 3-month induction of human ET-1 overexpression.

Methods:
Ten to 12-week-old male ieET-1 mice and control ieCre mice expressing a tamoxifen-inducible Cre recombinase under the control of endothelium-specific Tie2 promoter were treated with tamoxifen (1 mg/kg/day, s.c.) for 5 days and sacrificed 16 days or 3 months later. RNA was extracted from MAs of ieCre and ieET-1 mice for small and total RNA-sequencing using Illumina HiSeq-2500 and further studied using a systems biology approach. DE genes were identified with fold change >1.4 and
P
<0.005. DE genes were validated using another set of mice and type of vascular cells expressing them by reverse transcription-quantitative PCR (RT-qPCR).

Results:
No DE miR were detected, while DE genes were identified after 3-week (15↑ and 39↓) and 3-month exposures to human ET-1 overexpression (4↑and 3↓). RT-qPCR validated 3 of 7 3-month DE genes:
Khdrbs3
(KH domain containing, RNA binding, signal transduction associated 3),
Aqp1
(Aquaporin 1) and
Ly6e
(Lymphocyte antigen 6 complex locus E).
Khdrbs3
was increased and
Ly6e
decreased after 3-week and 3-month exposures to human ET-1 overexpression, while
Aqp1
was increased only after 3-month exposure. In mice,
Khdrbs3
and
Ly6e
were expressed in endothelial cells (ECs), smooth muscle cells (SMCs) and fibroblasts (FCs) and
Aqp1
was more expressed in ECs and FCs. In humans,
KHDRBS3
was expressed in the 3 vascular cell types,
LY6E
more in FCs, and
AQP1
more in ECs.

Conclusions:
Exposure for 3 weeks and 3 months to endothelial human ET-1 overexpression changed the expression of
Khdrbs3, Ly6e
and
Aqp1
in MAs. Vascular cells expressing these genes were identified. However, their role in ET-1-induced vascular injury remains to be determined.
Abstract P197: TRPM7 is Involved in the Effects of VEGF and EGF in Vascular Cells
Authors: Rios Francisco J , Zou ZhiGuo , Neves Karla B , Alves-Lopes Rheure , Camargo Livia L , Montezano Augusto C , Touyz Rhian M
Published: September 2019

TRPM6 and 7 are channels important in Mg
2+
and Ca
2+
homeostasis. We demonstrated that TRPM7 is influenced by angiotensin II. TRPM6 is influenced by EGF and cancer patients treated with EGFR inhibitors exhibit hypomagnesemia and hypertension by unclear mechanisms. Whether growth factors influence vascular TRPM7 is unknown. Here we questioned if VEGF/EGF mediate vascular effects through TRPM7. Studies were performed in human VSMC, wild type (WT) and TRPM7-deficient (M7+/Δ) mice. VSMC were stimulated with VEGF or EGF (50ng/ml) in the absence/presence of vatalanib, gefitinib (1μM), 2APB (30μM) and NS8593 (40μM), inhibitors of VEGFR, EGFR and TRPM7 respectively. Ca
2+
and Mg
2+
levels were assessed by Cal-520 and Mg-green. VEGF/EGF signaling was assessed by immunoblotting and vascular function by myography in mesenteric arteries from WT and M7+/Δ mice and treated with EGF or VEGF (50ng/ml). TRPM7 expression in aortas and kidneys from WT treated with vatalanib or gefitinib (100mg/Kg/day, 2 weeks) was assessed by immunoblotting. VEGF and EGF increased TRPM7 expression (50% and 67% respectively) and phosphorylation (2-fold), promoted influx of Ca
2+
(8% and 10%) and Mg
2+
(8%), effects that were reduced by vatalanib, gefitinib, NS8593, and 2-APB. EGF but not VEGF increased phosphorylation of PKC (43%), p38MAPK (47%), and ERK1/2 (120%). These responses were reduced by gefitinib, however only ERK1/2 phosphorylation was inhibited by NS8593, and 2-APB. Mice treated with vatalanib or gefitinib showed reduced expression of TRPM7 in aortas (50% and 74% respectively) and kidneys (36% and 66% respectively). Vessels exposed to EGF were less responsive to acetylcholine (ACh)-induced relaxation, [Emax %: WT (veh 97±3 vs EGF 63±10, p<0.05), M7+/Δ (veh 89%±5% vs EGF 69%±5%, p<0.05)]. Vessels from M7+/Δ treated with VEGF were less sensitive to sodium nitroprusside (SNP)-induced relaxation [pD2: WT (veh 7±0.12 vs VEGF 7.4±0.12, p<0.05), M7+/Δ (veh 6.7±0.15 vs VEGF 6.9±0.09)]. EGF and VEGF regulate VSMCs through TRPM7-dependent pathways. These processes involve MAP kinases and influence vascular function. Our findings identify novel mechanisms whereby growth factors influence vascular contraction/relaxation and suggest that TRPM7-regulated Mg
2+
and Ca
2+
are important.
Abstract P199: Gene Variants in Human Renal Proximal Tubule Alpha ENaC are Associated With Inverse Salt Sensitivity of Blood Pressure
Authors: Xu Peng , Sudarikova Charleston V , Ilatovskaya Daria V , Gildea John J , Yue Wei , Schiermeyer Katherine A , Felder Robin A
Published: September 2019

The amiloride-sensitive epithelial sodium channel (ENaC) localizes to the apical membrane of epithelial cells in distal nephron, playing an important role in regulating whole-body Na
+
homeostasis and blood pressure. We were the first to describe the presence of αENaC in human renal proximal tubule cells (hRPTC). Gene variants in αENaC have been shown to be associated with salt sensitivity of blood pressure
(SS)
. We also verified that the specific gene variant rs4764586 was significantly associated with SS (χ
2
=9.67, P=0.046). In our salt sensitivity clinical study, 11% participants (N=27/240) had a paradoxical increase in blood pressure (BP) (≥7-mm Hg) on a low NaCl diet, defined as inverse salt sensitive (
ISS
) as compared to salt resistant (
SR
, 72%) and salt sensitive (
SS
, 17%). The incidence of the minor allele αENaC rs4764586 in the
ISS
group was 2-fold of that in
SR
or
SS
(
ISS
, 14.8%;
SR
, 7.0%;
SS
, 7.1%). Expression of αENaC protein in urine derived hRPTCs was significantly lower in
ISS
than in
SR
(
ISS
, 0.55±0.01, n=3; SR, 0.97±0.12, n=3; SS, 0.87±0.12, n=3; one-way ANOVA,
ISS
vs
SR
p<0.05), with the lowest in the homozygous variants (HV) of rs4764586 and highest in wild type. The heterozygous variant containing hRPTCs had an intermediate expression of αENaC (WT, 0.94±0.11, n=4; Heterozygous, 0.72±0.11, n=4; HV, 0.53, n=1). ENaC-like channels were demonstrated in hRPTCs using single-channel patch-clamp electrophysiology with a conductance of 10.5±0.7 pS and E
rev
39.2±5.5 mV vs 10.3±0.8 pS in SR and ISS lines, respectively. However, in
ISS
baseline ENaC-like channel activity was too low to be recorded without trypsin in the pipette (Po = open probability), unlike
SR
. In contrast,
ISS
cells demonstrated a higher Po than
SR
cells in response to trypsin both immediately and after 4 minutes of activation, even though the number of channels recorded at 4 min after GigaOhm seal formation was lower in the ISS line compared to
SR
line (4.7±1.7 vs 1.75±0.5, p=0.042 in
SR
vs
ISS
lines). These results suggest that
ISS
phenotype may be related to impaired ENaC activity in the renal proximal tubule. The long-term physiologic consequences of the
ISS
phenotype remain to be determined
Abstract P2002: Loss of GSTM1 is Cardioprotective Against Ischemia-Reperfusion Injury
Authors: Wang Yves T , Nehrke Keith , Brookes Paul S , Le Thu H
Published: September 2019

GSTM1
encodes the glutathione S-transferase μ-1 (GSTM1) enzyme that belongs to a superfamily of phase II antioxidant enzymes. In humans, a common deletion variant of the
GSTM1
gene, the
GSTM1(0)
null allele, results in decreased GSTM1 enzymatic activity and is associated with higher levels of oxidative stress. The highly prevalent
GSTM1
deletion variant is associated with an increased risk of a variety of diseases, including chronic kidney disease, hypertension, and coronary artery disease. In myocardial infarction, cardiac reperfusion following ischemia causes additional irreversible damage, mediated by a large burst of ROS production by the electron transport chain. It has previously been shown that glutathione detoxifies ROS generated during ischemia-reperfusion (IR) injury, reducing damage. As such, we hypothesized that the loss of GSTM1 would increase the severity of acute IR injury in the heart. We generated a
Gstm1
knockout (

Gstm1
-/-

) mouse line on a 129S6 background. Male mice were used at 14-20 weeks of age. IR injury was assessed in an
ex vivo
Langendorff-perfused heart model with a 25 min period of ischemia. Compared to age-matched wild-type controls,

Gstm1
-/-

hearts showed increased functional recovery (rate-pressure product (RPP): 49.8±10.3% vs. 7.4±2.1%, p = 0.049) and reduced infarct size (15.8±8.1% vs. 46.1±5.2%, p = 0.044) after 60 min of reperfusion. Contrary to expectations, the loss of the antioxidant GSTM1 is cardioprotective in an acute IR injury model, suggesting an adaptive or compensatory upregulation of other antioxidants in the heart. Additional studies are needed to identify the mechanism that protects the heart from IR injury in GSTM1 deficiency.
Abstract P2006: Imbalance Between Collagen Type 1 and 3 in the Remodeling of the Extracellular Matrix Contributes to the Transition to Heart Failure
Authors: Prado Cibele , Amorin Vanessa , Léllis Janaína , Rizzi Elen , Ramos Simone , Meirelles Flávio
Published: September 2019

Background:
Hypertension causes cardiac hypertrophy, cardiac dysfunction and heart failure (HF). Despite the importance of cardiac hypertrophy as an independent risk factor for the development of HF, not all hypertrophied hearts will ultimately fail. Alterations in the extracellular matrix have also been shown to contribute to this process. This study aimed to investigate whether alterations in the levels of profibrotic proteins contribute to the transition of compensated cardiac hypertrophy to HF.

Methods:
Male rats were subjected to abdominal aorta constriction. After 90 days post surgery (dps), the hearts were collected and Western blot was performed to collagens type I and III, MMP-14, MMP-2, TIMP-2, Integrin and TGF-β. The interstitial collagen area was quantified. Cardiac function was evaluated using the Vevo 2100 ultrasound. Data were considered significant when p<0.05.

Results:
At 90 dps, 75% (30 of 40) presented hypertrophic hearts (HH) and 25% (10 of 40) hypertrophic+dilated hearts (HD) in the echocardiography. In addition, systolic (ejection fraction: 33.5%±1.3; fractional shortening: 21.90±4.3%) and diastolic functions (mitral E-wave velocity: 1279±127 mm/s and E/E' ratio: 68.14±16.78) were importantly reduced in the HD group when compared to HH. Although the interstitial collagen content in the left ventricle was not different between HH (3.63±0.40%) and HD (3.75±0.39%) groups, the amount of type III collagen increased (1.28±0.34 AU) while type I collagen showed significant reduction (0.76±0.16 AU) in the HD group when compared to HH (0.90±0.26 AU and 1.11±0.11 AU, respectively). Important increases were seen in the protein levels of TGF-B, integrin, MMP-14, MMP-2 and TIMP-2 in the HD and HH groups when compared to sham, but there is no difference between HD and HH groups.

Conclusion:
Although there is significant fibrosis in the hearts of animals after 90 dps, the imbalance between type I and III collagens associated with systolic and diastolic function could contribute to the transition to decompensated hypertrophy in this experimental model of cardiac hypertrophy.
Abstract P2013: Genomic Identification Of Key CircRNAs In Healthy And Dilated Cardiomyopathy Human Heart
Authors: Dong Kunzhe , He Xiangqin , Su Huabo , Xin Hongbo , Zhou Jiliang
Published: September 2019

Heart failure is a leading cause of morbidity and mortality. Dilated cardiomyopathy (DCM) is a primary cause of heart failure and sudden cardiac death worldwide. Therefore, it is important to identify factors that function in both normal and diseased hearts for a better understanding of etiology of DCM. CircRNA is a newly discovered class of RNA molecule and has been found to play a critical role in both physiological and pathological conditions. In this study, we systematically analyzed whole transcriptome data of five normal and five DCM human heart samples to identify putative key circRNAs and predict their potential underlying mechanisms. Our results revealed that many circRNAs produced from known cardiac diseases-related gene loci are dysregulated in DCM, such as circRNAs originated from
TTN
and
RYR2
. Furthermore, our analysis revealed 40 conserved circRNAs that are putatively important in normal or DCM human heart, including 14 circRNAs with potential to act as sponges for DCM-related miRNAs and 4 circRNAs with potential coding ability. Importantly, our results identified that a novel class of circRNA, fusion-circRNAs are originated from
MYH6
and
MYH7
genes and exhibit most abundant expression in human heart with high conservation between different species. Furthermore, these fusion-circRNAs were dysregulated in DCM and have strong potential to sponge DCM-related miRNAs. In summary, our findings serve as a valuable resource for exploration of the function of circRNAs in human heart diseases.
Abstract P2015: Antiproliferative Effects of Monoclonal Antibodies Against (Pro)Renin Receptor in Pancreatic Ductal Adenocarcinoma
Authors: Nishiyama Akira , Matsuyama Makoto , Shibayama Yuki , Ebihara Akio , Rahman Asadur
Published: September 2019

We previously reportedthat silencing of the PRR gene, which encodes the(pro)renin receptor ((P)RR) significantly reduced Wnt/β-catenin-dependent development of pancreatic ductal adenocarcinoma (PDAC). Here, we examined the effects of a panel of blocking monoclonal antibodies (mAbs) directed against the (P)RR extracellular domain on proliferation of the human PDAC cell lines PK-1 and PANC-1 in vitro andin vivo. We observed that four rat anti-(P)RR mAbs induced accumulation of cells in the G0/G1 phase of the cell cycle and significantly reduced proliferation in vitroconcomitant with a significant reduction in the expression of active β-catenin and cyclin D1. Systemic administration of the anti-(P)RR mAbs to nude mice bearing subcutaneous PK-1 xenografts significantly decreased tumor expression of active β-catenin and the proliferation marker Ki-67 and reduced tumor growth. In contreffects ast, treatment with the handle region peptide of (pro)renin did not inhibit tumor growth in vitro orin vivo, indicating that the effects of the anti-(P)RR mAbs was independent of the renin-angiotensin system. These data indicatethat mAbs against human (P)RR can suppress PDAC cells proliferation by hindering activation of the Wnt/β-catenin signaling pathway. Thus, mAb-mediated (P)RR blockade could be an attractive therapeutic strategy for PDAC.
Abstract P2021: Cutaneous Wound Healing in Diabetic Mice is Improved by Topical Mineralocorticoid Receptor Blockade
Authors: Nguyen Van Tuan , Farman Nicolette , Aractingi Selim , Jaisser Frederic
Published: September 2019

Introduction:
Chronic skin ulcers resulting from impaired wound healing are a serious complication of diabetes. Unresolved inflammation, associated with the dysregulation of both the phenotype and function of macrophages, is involved in the poor healing of diabetic wounds.

Objective:
To investigate the role of the mineralocorticoid receptor in the healing delay and specifically in the impaired angiogenesis and prolonged inflammation of diabetic wounds.

Methods:
6 mm wounds were created on the back skin of female streptozotocin (STZ)-induced or db/db mice. Wounds were treated topically with canrenoate (Canre: 0.5mM) or PBS twice a day, for 5 (STZ mice) or 7 (db/db mice) days. Wound area was measured at indicated time post-wounding. Wounded skin was collected for histology and molecular analysis.

Results:
topical pharmacological inhibition of the MR by Canre can resolve prolonged inflammation to improve delayed skin wound healing in diabetic mouse models. The improvement is demonstrated by increasing of 39% of neo-epidermisation and 62% of epidermal keratinocyte proliferation in db/db wounds treated by Canre. Moreover, Canre could improve the reduced blood vessel density in the wound beds of STZ mice (STZ + PBS: 5.33% ± 0.32 vs STZ + Canre: 8.26% ± 0.64) and of db/db mice (db/db + PBS: 1.32% ± 0.29 vs db/db + Canre: 3.31% ± 0.33). The beneficial effect of Canre is associated with an increased ratio of anti-inflammatory M2 macrophages to pro-inflammatory M1 macrophages in diabetic wounds (ratio of M2 to M1 in db/db + PBS vs db/db + Canre: 0.72 ± 0.15 vs 1.72 ± 0.29, respectively). Furthermore, we show that MR blockade leads to downregulated expression of a MR target, lipocalin 2 (Lcn2), which may facilitate macrophage polarization towards the M2 phenotype and promote impaired angiogenesis in diabetic wounds. Indeed, diabetic Lcn2-deficient mice showed improved wound healing, associated with macrophage M2 polarization and angiogenesis. In addition, recombinant Lcn2 protein prevented IL4-induced macrophages switch from M1 to M2 phenotype.

Conclusion:
topical MR blockade accelerates skin wound healing in diabetic mice via Lcn2 reduction, M2 macrophage polarization, prevention of prolonged inflammation, and induction of angiogenesis.
Abstract P2026: Involvement of Subfornical Organ Insulin Receptor Signaling in the Development of Angiotensin-II Hypertension
Authors: Jeong Jin Kwon , Simonyan Hayk , Young Colin
Published: September 2019

Neurocardiovascular dysfunction due to angiotensin-II (Ang-II) signaling within the brain has emerged as a key mechanism in hypertension development. Interestingly, the hormone insulin is also closely associated with multiple forms of hypertension. Emerging evidence suggests interactions between insulin receptor and Ang-II type 1a receptor (AT
1a
R) signaling, although the extent to which this contributes to hypertension development remains unclear. Based on this, we hypothesized that brain insulin receptor signaling is involved in Ang-II hypertension. Male C57Bl/6J mice were instrumented with intracerebroventricular cannula (ICV) and radiotelemeters for conscious cardiovascular measurements. Subsequently, administration of Ang-II (600 ng/kg/min) over 2 wk resulted in the development of hypertension in control animals; daily ICV insulin receptor antagonism with the pharmacological agent S961 (3.5 nM) prevented hypertension development (e.g. day 14 mean arterial pressure 118±6 vs 110±6 mmHg, ICV vehicle vs ICV S961, p=0.05). Given the well-known role for the subfornical organ, a forebrain region that lacks a blood-brain-barrier, in this model of hypertension, we next focused our attention on this nucleus. Insulin receptor immunohistochemistry in AT
1a
R eGFP reporter mice (GENSAT NZ44) revealed that insulin receptors were present on 69% of AT
1a
R cells (42 of 62, n=3) in the SFO. Acute insulin administration (i.p. 5U) was also used to explore SFO insulin receptor-associated intracellular signaling including phosphorylation of AKT [pAkt; phosphatidylinositol 3-kinase (PI3K) signaling] and ERK1/2 [pERK; mitogen-activated protein kinase (MAPK) signaling]. Analysis of over 4000 SFO cells indicated that insulin resulted in activation of the PI3K pathway as demonstrated by an increase in the percentage of cells with medium to high pAKT expression (5±1 vs 14±1% per section, vehicle vs insulin, n=3-4, 5-10 sections/animal). In contrast, acute insulin administration did not influence SFO pERK. Collectively, these findings indicate that brain insulin receptor signaling is necessary for the development of Ang-II hypertension, and that this response may be mediated by interactions between Ang-II and insulin and/or PI3K signaling in the SFO.
Abstract P2032: Differential Response To Angiotensin II Depends On Microbial Colonization
Authors: Avery Ellen G , Bartolomaeus Hendrik , Kraeker Kristin , Balogh András , Müller Dominik N , Wilck Nicola
Published: September 2019

The gut microbiome is suspected to play a role in hypertension and target organ damage. In the present study, we used the germ-free mice (GF), which are devoid of bacteria, to investigate if bacterial colonization modulates the response to a hypertensive stimulus.

GF C57BL/6 littermates were separated at 4 weeks and randomized to remain GF or receive bacterial colonization (COL). Angiotensin (Ang) II minipumps were implanted at 12 weeks (+AngII) to induce hypertension, sham-infused mice served as non-hypertensive controls. At 14 weeks, all mice were sacrificed, and inflammation and target organ damage were assessed.

AngII induced a comparable cardiac hypertrophic response in both groups, as measured by left ventricular mass/tibia length. However, accounting for the low basal heart weight in non-hypertensive GF, the hypertrophic response was greater in GF than COL (in mg/cm; GF: 47.2, GF+AngII: 70.17, fold change: 1.5 and COL: 57.9, COL+AngII: 76.09, fold change: 1.3). Similarly, the increase in cardiac perivascular fibrosis in response to AngII was greater in GF than COL (fibrotic/medial area; GF: 3.8, GF+AngII: 5.8 and COL: 3.9, COL+AngII: 5). Inflammation of the cardiac tissue, as evidenced by relative mRNA expression of MCP-1 and TNFa, increased significantly in GF and not in COL. Using flow cytometry, we found that innate anti-inflammatory immune subsets (myeloid derived suppressor cells) are notably missing in GF, and the adaptive immune response to AngII (e.g. Th17 (in % of CD4+: GF: 0.26; GF+AngII: 0.5 and COL: 0.28; COL+AngII: 0.39)) was exacerbated. Kidney damage was also evident in AngII-infused GF and COL. However, genes indicative of tubular damage (KIM-1: GF: 0.3, GF+AngII: 3.3, fold change: 11.0 and COL: 0.2, COL+AngII: 1.5, fold change: 7.5 ), inflammation (TNFa, MCP-1, ICAM), and fibrosis (Col3a1, Col1a2) increased significantly in GF and were only mildly implicated in COL.

Bacterial colonization sizably impacts the response to AngII and hypertensive target organ damage. The magnitude of the pathogenic response to AngII in GF compared to COL mice demonstrates the importance of the gut microbiota in modulating target-organ damage.
Abstract P2033: Rationale For A Novel Approach To Hypertension Treatment Based On Immuno-neutralization Of Angiotensin-(1-12) Catalytic Sites With A Polyclonal Antibody
Authors: Ahmad Sarfaraz , Wright Kendra N , Varagic Jasmina , Groban Leanne , Ferrario Carlos M
Published: September 2019

The biochemical pathways for the formation of biological active angiotensins continues to undergo significant revision with the demonstration of angiotensin-(1-12) as an endogenous substrate forming angiotensin II (Ang II) by a non-renin dependent mechanism. Research done in our laboratory established that Ang II generation from Ang-(1-12) is primarily mediated by ACE in the circulation and by chymase in cardiac tissues. In this study, we have utilized an antigen-antibody binding approach to block the cleavage sites of human Ang-(1-12) peptide with an anti-Ang-(1-12) polyclonal antibody (pAb) as a potential therapeutic strategy to prevent the catalytic action of chymase and ACE enzymes to generate Ang II. The pAb was generated against the C-terminus of the human Ang-(1-12) peptide sequence and its specificity for human Ang-(1-12) and cross-reactivity with other angiotensin peptides were determined in competitive binding assays. To block the cleavage sites, the radiolabeled human Ang-(1-12) [
125
I-Ang-(1-12)] substrate was first neutralized by pre-incubating with or without anti-Ang-(1-12) pAb and then re-incubated with human recombinant chymase (hrChymase, 0.325 μg/mL) or 10 μL of rat serum (for ACE) in 50 mM Tris-HCl buffer solution containing 150 mM NaCl (pH 8.0) for 30 min at 37
o
C. The reaction was stopped by adding an equal volume of 1% phosphoric acid and
125
I-Ang metabolic products were quantified by HPLC connected to an in-line flow-through gamma detector. Anti-Ang-(1-12) pAb showed high specificity for the human Ang-(1-12) sequence (EC
50
= 29.6 ng/mL) and negligible cross-reactivity with closely related angiotensin peptides. The non-neutralized human
125
I-Ang-(1-12) substrate was rapidly metabolized by both hrChymase and rat serum ACE to
125
I-Ang II (81% and 35%, respectively). In contrast, generation of
125
I-Ang II from neutralized
125
I-Ang-(1-12) substrate by either hrChymase or ACE was only 17% and 7%, respectively. The effective and potent blockade of Ang II generation from either chymase or ACE by combining Ang-(1-12) with an anti-Ang-(1-12) pAb now paves the way to develop new therapeutic approaches to hypertension treatment using Ang-(1-12) antibodies directed against the human sequence of this Ang II-forming substrate.
Abstract P2034: Effects of Renal Denervation on the Course of Heart Failure and Occurrence of Renal Dysfunction in Ren-2 Transgenic Hypertensive Rats With Aorto-Caval Fistula
Authors: Honetschlagerova Zuzana , Cervenka Ludek
Published: September 2019

Objective:
We hypothesized that renal denervation (RDN) attenuates the progression of aorto-caval fistula (ACF)-induced heart failure (HF) and the development of renal dysfunction in Ren-2 transgenic rats (TGR), a model of ANG II-dependent hypertension.

Methods:
RDN (severing renal nerve fibers, followed by phenol application) was done one week after creation of ACF; the follow-up period was 70 days. The animals studied were: 1) Sham-operated TGR (initial n = 8); 2) Sham-operated TGR + RDN (n = 9); 3) ACF TGR (n = 31); 4) ACF TGR + RDN (n = 34). In separate groups (n = 8 in each), three weeks after induction of ACF, renal artery blood flow (RBF, Transonic probe) responses were determined to intrarenal ANG II (2 and 8 ng), norepinephrine (NE) (20 and 40 ng) and acetylcholine (Ach) (10 and 40 ng).

Results:
Untreated ACF TGR began to die 3 weeks after induction of ACF and the final survival rate was 10% (3 from 31). RDN substantially improved the final survival rate to 50% (17 from 34). Basal RBF was significantly lower in ACF TGR than in sham-operated TGR (6.9 ± 0.4 vs. 10.1 ± 0.5 ml.min
-1
.g
-1
, p<0.05) and RDN did not alter it in any group. Both doses of ANG II decreased RBF more in ACF TGR than in sham-operated TGR (-17 ± 2% vs. -9 ± 2% and -42 ± 3% vs. -25 ± 2%, p<0.05 in both cases) and RDN increased RBF responsiveness to ANG II in ACF TGR (to -29 ± 3% and to -74 ± 3%, p<0.05 in both cases) as well as in sham-operated TGR (to -21 ± 3% and -42 ± 3%, p<0.05). Intrarenal NE comparably decreased RBF in ACF TGR and sham-operated TGR, and RDN increased RBF responsiveness in both groups. Intrarenal Ach increased RBF significantly more in ACF TGR than in sham-operated TGR (+24 ± 2% vs. +11 ± 2% and +26 ± 2% vs. +13 ± 2%, p<0.05 in both cases) and RDN did not alter it in any group.

Conclusion:
The results show that RDN significantly improved survival rate in ACF TGR, however this beneficial effect was not associated with improvement of reduced RBF or with attenuation of exaggerated renal vascular responsiveness to ANG II.
Abstract P2039: Nitric Oxide Inhibition Enhances the Protein Expression of TNF-α Receptor Type 1 in Cultured M-1 Cells With Characteristics of Renal Collecting Duct Epithelium
Authors: Castillo Alexander , Talwar Sudha , Abdel-Mageed Asim B , Ekpo Peace E , Majid Dewan S
Published: September 2019

Oxidative stress and inflammation are key factors in the development of salt sensitive hypertension (SSH). Enhanced sodium reabsorption in the distal nephron, particularly the collecting duct (CD) plays a critical role in SSH development. Although the decrease in nitric oxide (NO) production (oxidative stress) and the increase in tumor necrosis factor-alpha (TNFα; pro-inflammatory cytokine) are common factors involved in SSH, the interactive role of these factors in modulating sodium reabsorption in CD cell is not yet clearly understood. Activation of TNF-α receptor type 1 (TNFR1) induces natriuresis by inhibiting ENaC, which is abundant in renal CD cells. The present study examined the effect of NO inhibition on TNFR1 protein expression in cultured M-1 cells (ATCC® CRL-2038™) that maintain the characteristics of renal cortical collecting duct cells. These cells were grown in DMEM-F12 growth medium containing 10% FBS and 1% Penstrep at 37
0
C, 5% CO2, 99% relative humidity up to 90% confluence. Then these cells were trypsinized and seeded into 100mm petri-dishes and treated with NO inhibitor, Nitro-L-arginine methyl ester (L-NAME; 0.5 mM) alone and in combination with a NO donor compound (DETA NONOate; 0.5 mM) along with a vehicle (PBS) treated group for 24 hours followed by harvesting and lysed with protease inhibitor-containing RIPA buffer. Extracted Protein concentration was determined by BCA assay kit, and a predetermined protein concentration was loaded in each well for SDS-PAGE. Immunoblots were detected for TNFR1 using primary antibodies for TNFR1 (19139; Abcam) and GAPDH. It was observed that TNFR1 expression significantly increases to 68±9.9 % (n=6) in L-NAME treated cells compared to vehicle treated control cells (n=4). This increase was prevented in cells co-treated with DETA NONOate (n=6). Immunofluorescence staining for TNFR1 protein in these treated cells also showed qualitatively similar findings as in western blot analysis. As TNFR1 mediates TNFα induced natriuretic response in the kidney, these data suggest that enhanced TNFR1 activity during NO inhibition in CD cells helps to minimize excessive sodium retention, and thus plays a counter-regulatory role in the development of SSH induced in oxidative stress conditions.
Abstract P2041: Wnt Signaling Regulates GPR37l1 Expression in Human Renal Proximal Tubule Cells
Authors: Armando Ines , Jose Pedro A , Konkalmatt Prasad R
Published: September 2019

Aberrations in renal epithelial ion transport contribute to the pathogenesis of essential hypertension. G protein-coupled receptors (GPCRs) in the kidney regulate the reabsorption of essential nutrients, ions, and water from the glomerular filtrate. The functional role and regulation of expression of several GPCRs in the kidney have yet to be explored. Recently we reported that G protein-coupled receptor 37L1 (GPR37L1) is expressed in the apical membrane of renal proximal tubule cells (RPTCs) and participates in luminal sodium transport and blood pressure homeostasis via the regulation of NHE3 expression. However, how GPR37L1 expression is regulated in RPTCs has not been studied. In silico analyses of GPR37l1 promoter revealed the presence of T-cell factor/lymphoid enhancer factor (TCF/LEF) transcription binding sites and suggesting that Wnt signaling, via the canonical beta-catenin (βCτνν) pathway, may positively regulate the expression of GPR37L1. To test the effect of Wnt signaling on the expression of GPR37L1, we treated hRPTCs with the activators of Wnt signaling (lithium chloride (LiCl) 0, 5, 10, 20, 25, 30 mM for 0, 3, 6, 9, 15, or 24 hr or recombinant Wnt3a (rWnt3a), 100 ng/ml/24 hr). The expressions of GPR37L1 and Wnt signaling mediators were analyzed by real-time RT-qPCR and immunoblotting. We found that activation of Wnt signaling by LiCl and rWnt3a decreased the expression of GPR37L1 mRNA in a time- and concentration-dependent manner. The expression of Cyclin D1 (CCND1), one of the Wnt signaling target genes, was increased in a time- and concentration-dependent manner. LiCl (25 mM/24 hr) or rWnt3a (100 ng/ml/24 hr) decreased GPR37L1 protein by 68±14% and 45±6%, respectively, but increased CCND1 expression 1.8±0.16-fold and 1.62±0.12-fold, respectively. The activation of Wnt signaling in hRPTCs was confirmed by the phosphorylation status of the mediators of Wnt signaling, βCTNN and glycogen synthase kinase 3 beta (GSK3β). LiCl decreased the ratio of phospho-βCTNN to total-βCTNN by 48±5% and increased the phospho-GSK3β to total-GSK3β by 2.65±0.25-fold. These results show that activation of Wnt signaling negatively regulates the expression of GPR37L1 in hRPTCs.
Abstract P2044: Renoprotective Effects of Canagliflozin in Db/db Diabetic Mice
Authors: Thanekar Unmesha , Gill Rupinder K , Dhakal Sanjeev , Hosawi Anhar , Elased Khalid M
Published: September 2019

Diabetes is one of the major causes of chronic kidney disease which could lead to end-stage renal disease. Previously, we have shown increased urinary angiotensin converting enzyme 2 (ACE2) and neprilysin (NEP) in
db/db
mouse model of type 2 diabetes, which was attenuated by normalizing hyperglycemia and glycosuria with the PPAR-γ agonist, rosiglitazone and pioglitazone. Sodium glucose co-transporter 2 (SGLT-2) inhibitor controls hyperglycemia by inhibiting renal glucose reabsorption and cause glycosuria. The aim of this study is to investigate the effect of SGLT-2 inhibitor, Canagliflozin on the urinary ACE2 and NEP and also to determine its effects on albuminuria and some of the renal injury biomarkers, neutrophil-gelatinase associated lipocalin (NGAL), sirtuins-1 (SIRT1) and arginase-II (Arg-II). Six- weeks old
db/db
and lean control mice were fed either normal chow or canagliflozin diet (20mg/kg/day) for 15 weeks. Metabolic and renal parameters were measured weekly and urine was collected for evaluating glycosuria, protein expression and enzyme activity. At 5 weeks,
db/db
mice showed significant increase in urinary ACE2, NEP and NGAL expression compared to lean mice (p<0.001). Treatment with canagliflozin for 15 weeks significantly decreased blood glucose in
db/db
compared to untreated mice (206.58 ± 38.35 vs. 461.82 ± 40.63 mg/dL,
p
<0.0001), with no effect in lean mice. Unlike PPAR-γ agonist, management of hyperglycemia with canagliflozin was not associated with attenuation of urinary albumin and ACE2. However, it attenuated urinary NGAL expression. As expected canagliflozin increased renal glycosuria in
db/db
and lean mice (
p
<0.0001) which was associated with significant increase in urinary NEP expression and activity. Western blot showed significant downregulation of SIRT1 in the kidney of 21 weeks old
db/db
mice which was upregulated by canagliflozin treatment. Further, there was upregulation of renal Arg-II in the
db/db
mice which was downregulated by canagliflozin. In conclusion, the renoprotective effects of canagliflozin could be mediated by upregulation of renal SIRT1 expression and downregulation of the renal injury markers, NGAL and Arg-II.