bluuebunny/crossref_metadata_2025 huggingface dataset:
Role of the Bradykinin B
2
Receptor in Salt-Induced Hypertension
Authors: Duka Irena , Johns Conrado , Gavras Irene , Bresnahan Margaret R , Gavras Haralambos
Published: March 2022

P189

The kallikrein-kinin system may contribute to blood pressure (BP) regulation via its vasodilatory, natriuretic and diuretic activity. In order to explore the role of bradykinin in this respect, we studied knockout mice (n=16) with deleted gene for the B
2
receptor of bradykinin (BKB
2
R -/-), submitted to subtotal nephrectomy (SN) and dietary salt-loading, in comparison to their wildtype counterparts (BKB
2
R +/+, n=10). Systolic BP mmHg (by tail cuff), heart rate (HR b/min), plasma catecholamines, (NEPI, EPI ng/ml) and heart weight/body weight ratio (mg/g) are shown in the Table below. *p<0.05 knockouts vs wildtype. Thus, the BKB
2
R

gene knockout mice had higher BP at baseline and developed more severe hypertension. Moreover, they became hypertensive in 19±2 days versus 28±2 days for the wildtype (p<0.05). We conclude that bradykinin, acting via its B
2
receptor, contributes to maintenance of normotension and protects against salt-induced hypertension.
Angiotensin-(1-7) Produces Hypotensive Responses in Anesthetized At
1a
Receptor Knockout Mice [Agtr1a(-/-)].
Authors: Callahan Michael F , Smith Thomas L , Oliverio Michael I , Coffman Thomas M , Chappell Mark C , Averill David B , Ferrario Carlos M , Diz Debra I
Published: March 2022

P193

Angiotensin-(1-7) [Ang-(1-7)] produces arteriolar dilation and a hypotensive action, which may be mediated through nitric oxide, prostaglandin or bradykinin mechanisms. In rats, Ang-(1-7) produces a biphasic pressor/depressor action; the pressor component is blocked by AT
1
antagonists while the depressor component is not blocked by either AT
1
or AT
2
antagonists. In this study, we determined whether Ang-(1-7) would produce cardiovascular effects in AT
1a
receptor knockout mice. Male Agtr1a(-/-) (n = 5) and control mice (n = 6) were anesthetized with ketamine:xylazine (71:7 mg/kg) and received femoral artery and jugular vein catheters. As previously reported, baseline blood pressure was lower (62 ± 5 mm Hg) in the Agtr1a(-/-) than in the controls (79 ± 6 mm Hg). Arterial blood pressure responses to Ang-(1-7) (0.15 - 1.5 μmol/kg) were determined. Ang-(1-7) produced a dose dependent pressor response in control animals with the peak response at the highest dose (15 ± 2 mm Hg). The pressor response was followed immediately by a depressor response ranging from -6 ± 1 at the lowest dose to -8 ± 1 mm Hg for the highest dose. AT
1a
receptor knockout mice showed no pressor response to Ang-(1-7) but did show an immediate depressor response ranging from -4 ± 1 to -6 ± 2 mm Hg. Similar reductions in blood pressure without pressor effects were observed in female knockout mice (-5 ± 2 mm Hg to -9 ± 2 mm Hg over the same dose range, n = 3). The results of this study indicate that the depressor response to Ang-(1-7) is not dependent on a preceding pressor response to this peptide. Furthermore, the depressor response is not mediated by an AT
1a
receptor, consistent with our previous reports of a non-AT
1
, non-AT
2
receptor mediating the actions of Ang-(1-7) in the vasculature. (Support: HL51952; HL55082)
Angiotensin Paradoxically Upregulates a Human Renin Transgene Containing a 900bp Promoter by a Pressure-Independent Mechanism
Authors: Keen Henry L , Sigmund Curt D
Published: March 2022

P196

Renin gene expression is negatively regulated by angiotensin II (AII) and blood pressure (BP). In transgenic mice containing the entire human renin (HREN) gene with only 900 bp of promoter sequence, pressor AII infusion paradoxically increases renal HREN mRNA. Lack of upregulation during subpressor AII suggests that this increase might be mediated by increased BP. To test this hypothesis, the AII-induced increase in BP was prevented by coadministration of the vasodilator, hydralazine (H). AII and H were infused at 1000 ng/kg/min and 15 mg/kg/day, respectively, by osmotic minipump. Isotonic saline was infused as vehicle (V). BP was measured daily by tail cuff, after a 7-day training period and a 5-day baseline period. Renal renin mRNA expression was measured by ribonuclease protection assay (normalized to expression of actin). BP during the last 3 days of a 5 day infusion of AII (n=5) was increased by an average of 37 ± 4 mmHg. There was no significant change in BP in mice infused with V (n=4) or with AII+H (n=4). The level of endogenous mouse renin mRNA was appropriately decreased by about 60% in both AII and AII+H mice relative to V mice. In contrast, HREN mRNA increased almost 2-fold (1.9 ± 0.3) in AII mice compared to V mice. In addition, the normalization of BP in AII+H mice did not prevent the increase in HREN mRNA (2.1 ± 0.3 fold). Thus, renal HREN in transgenic mice containing the entire HREN gene with 900 bp of promoter sequence is increased by pressor AII even when the BP increase is ameliorated by H, suggesting that the upregulation may be due to a direct effect of AII. Moreover, the finding from our previous study of a lack of upregulation during subpressor AII infusion may indicate that this action of AII is dose-dependent. We are currently performing similar studies in transgenic mice containing a well regulated HREN transgene to further elucidate the molecular mechanisms of renin gene regulation and to better understand the defect existing in the present model.
Abstract 107: Home Electronic Blood Pressure Cuff Improved Compliance with Disease-Specific as well as General Blood Pressure Goals in an Underserved High-Risk Ambulatory Population
Authors: Zalkind Daniel
Published: March 2022

Objective:
To determine the effectiveness of home electronic blood pressure cuffs to improve compliance with both disease-specific and the general Practice Performance Measures (PCPI) blood pressure goals in the high risk underserved population in a safety-net urban medical center.

Background:
Achieving recommended blood pressure goals in an underserved patient population with a high cardiac disease burden is challenging. Intensive, clinician-led health maintenance programs have shown promise but are difficult to implement on a wide scale. The decreasing cost of electronic blood pressure cuffs offers an opportunity to provide patient-focused treatment.

Methods:
Twenty-two consecutive patients who were not at either their disease-specific or PCPI goal of <140/90mmHg received an electronic blood pressure cuff for home use as part of their optimal medical treatment. This was a prospective, before-after study. Patients were relatively young (mean 59 years), had a high prevalence of coronary disease (32%), heart failure (37%) , valvular disease(37%) and preserved left ventricular ejection fraction (49%). In-clinic blood pressure measurements were collected. The time to the cross-over from medical therapy to medical therapy with a blood pressure cuff was an average 184.4 days (3.2 encounters with clinicians for medication changes) and average of 75.8 days (1.1 encounters) after the cross-over to follow-up. A paired student T-test was used for statistical comparison between the two groups.

Results:
There was a statistically significant difference in the systolic blood pressures after the medical treatment period compared to the cross-over arm 143.3mmHg (CI 135.3-151 p-value=0.1) vs.134.6mmHg and diastolic pressure 81.09mmHg (CI 75.4-86,75 p-value=0.05) vs. 72.24mmHg. Overall compliance with the PCPI recommendation was higher in the cross-over arm (45% vs. 55%) as well as with the disease specific recommendations (18% vs. 25%).

Conclusion:
There was a statistically significant improvement in both systolic and diastolic blood pressures in the underserved ambulatory patient with a home blood pressure cuff. There was also an improvement in compliance both with the PCPI and disease-specific goals guidelines.
Abstract 117: Contribution of Endocannabinoids via CB1 Receptors to the Impaired Baroreflex Sensitivity in (mRen2)27 Transgenic Hypertensive Rats
Authors: Schaich Chris L , Howlett Allyn C , Diz Debra I
Published: March 2022

There is growing evidence for a role of the endocannabinoid system in the pathogenesis of hypertension and associated metabolic syndrome. Presynaptic CB1 cannabinoid receptors, the neuronal target of endocannabinoids, are densely expressed in the solitary tract nucleus (NTS), the primary site for termination of baroreceptor afferent nerve fibers, and in vagal afferent neurons of the nodose ganglion. Our studies in anesthetized, normotensive Sprague-Dawley rats suggest that activation of cannabinoid receptors within the NTS via bilateral microinjection of the CB1 agonist CP55,940 modulates baroreflex sensitivity (BRS) for control of heart rate (HR) in a dose-related biphasic manner. Administration of the CB1-selective antagonist SR141716A (36 pmol) fully prevented or reversed these changes in BRS, and there was no significant effect of the antagonist alone in these normotensive animals (1.04 ± 0.05 ms/mmHg baseline vs. 1.12 ± 0.11 ms/mmHg after antagonist treatment, n = 5). We have now determined the effects of blockade of CB1 receptors in a model of angiotensin (Ang) II-dependent hypertension and insulin resistance with impaired BRS. In contrast to observations in normotensive rats, SR141716A dose-dependently improved BRS for control of HR when microinjected into the NTS of hypertensive, transgenic (mRen2)27 rats with impaired baseline BRS, without changing resting MAP or HR. Microinjection of 0.36 pmol SR141716A (n = 5) increased BRS from a baseline of 0.43 ± 0.03 ms/mmHg to 0.68 ± 0.02 (p < 0.001), and 36 pmol SR141716A (n = 5) increased BRS from 0.47 ± 0.02 to 1.05 ± 0.07 ms/mmHg (p < 0.001), a level that was comparable with baseline BRS of normotensive Sprague-Dawley rats. The demonstration of dose-dependent actions of the CB1 agonist on BRS in normotensive animals coupled with new findings that blockade of CB1 receptors improves BRS, an index of vagal function, in (mRen2)27 hypertensive animals is consistent with the interpretation that elevated endocannabinoids within the NTS contribute to impaired BRS. Thus, our findings suggest that blockade of CB1 receptors will influence cardiovagal reflexes in a positive manner in the context of hypertension and metabolic syndrome.
Abstract 125: Elevated Cd73-mediated Increased Renal Adenosine Signaling Contributes to Chronic Hypertensive Nephropathy via A2b Adenosine Receptor
Authors: Zhang Weiru , Wang Wei , Dai Yingbo , Zhang Yujin , Ning Chen , Grenz Almut , Zhang Wenzheng , Tao Lijian , Sun Hong , Blackburn Michael , Eltzschig Holger , Kellems Rodney , Blackburn Michael , Xia Yang
Published: March 2022

Hypertension is the most prevalent life-threatening disease worldwide and is frequently associated with chronic kidney disease (CKD). However, the molecular basis underlying hypertensive CKD is not fully understood. Here we sought to identify specific factors and signaling pathways that contribute to hypertensive CKD and thereby exacerbate disease progression. Using non-biased high throughput quantitative RT-PCR profiling, we discovered that the expression level of 5’-ectonucleotidase (CD73), a key enzyme that produces extracellular adenosine, was significantly increased in the kidneys of angiotensin II (Ang II)-infused mice, a well-accepted animal model of hypertensive nephropathy. Similarly, in human, we found that CD73 levels were significantly elevated in the kidneys of CKD patients compared to normal individuals and further elevated in hypertensive CKD patients. Next, we provide both genetic and pharmacological evidence that elevated CD73 contributes to Ang II-induced hypertension, kidney injury and progression to renal fibrosis by chronic elevation of adenosine locally in the kidney (from 1.275±0.44 to 4.278±0.84 nmole/mg protein), but not systemically in the circulation. Mechanistically, we showed that excess renal adenosine preferentially activated the A
2B
adenosine receptor (ADORA2B) and that selective inhibition of the ADORA2B or genetic deficiency of the ADORA2B significantly reduces hypertension (168±12 to 120±15 mmHg) and proteinuria (75±14 to 25±5 μg albumin/mg creatinine), while increasing urinary osmolarity (780±105 to 2145±156, all
P
<0.05) and halts progression to renal fibrosis in Ang II-infused mice. These findings led us to further discover that adenosine signaling via ADORA2B activation stimulates kidney production of endothelin-1, a potent vasoconstrictor, in a HIF-1α-dependent manner and underlies the pathogenesis of the disease. Overall, our studies reveal that CD73-mediated chronic elevation of renal adenosine is a prominent driver of hypertensive CKD and inhibition of excess adenosine-mediated ADORA2B signaling is a novel therapeutic target for the disease.
Abstract 139: Doca-salt Induced Vascular Dysfunction and Elevated Arginase Activity is Prevented in Haploinsufficient Rho Kinase 1 Mice
Authors: yao lin , Toque Haroldo , Nunes Kenia , Webb R. Clinton , Caldwell Ruth , Caldwell R. William
Published: March 2022

Endothelial dysfunction is an established phenomenon in deoxycorticosterone acetate (DOCA)-salt hypertension. It has been reported that increased vascular arginase activity is involved with hypertension. We previously reported that the small GTPase, RhoA and Rho kinase (ROCK) are involved with activation of arginase in diabetic and angiotensin II hypertensive models. In this study, we hypothesized that partial knockdown of ROCK1 would prevent vascular endothelial dysfunction in DOCA-salt hypertension by blocking the rise in vascular arginase activity. We tested this concept using wild type and haploinsufficient ROCK1 knockout (R1
+/-
) mice, which were treated by DOCA pellet (50 mg) for 6 weeks and fed by 1% saline. Systolic blood pressure (SBP) was measured before and during 6 week treatment from wild type sham (WT), wild type DOCA (WT-D), ROCK1 sham (R1
+/-
) and ROCK1 DOCA (R1
+/-
-D) mice (N = 6). SBP in WT-D mice rose over the 6 weeks treatment (from 104.6.6±2.6 mmHg to 124.4±5.6 mmHg) but not in WT mice (from 108.9±1.2 mmHg to 104.60±4.27 mmHg, p<0.05). At 6 weeks, SBP in R1
+/-
-D mice (108.4±2.8 mmHg) was slightly higher than R1
+/-
mice (98.9±1.6 mmHg, p<0.05). However, SBP in R1
+/-
-D mice is significantly lower than WT-D mice during the whole treatment period. Additionally, vascular function was assessed in aortic segments (N = 4-6). We found WT-D mice exhibited a marked reduction of maximum endothelium-dependent relaxation (MER) to acetylcholine (57.6±4.3%, p<0.05) compared to WT mice (70.6±1.6%). Impairment of relaxation was absent in R1
+/-
-D mice (MER: 82.9±2.8%, p<0.05). Arginase activity in aorta of WT-D mice (152.7±18.7 units) was ∼45% higher than in WT mice (105.3±2.2 units) and ∼225% higher than in R1
+/-
-D mice (67.1±5.8 units). These data demonstrate involvement of ROCK1 in enhanced arginase activity and impaired endothelium dependent vasorelaxation in DOCA-salt hypertensive mice.
Abstract 14: T Cells Mediate Angiotensin II-induced Aortic Stiffening
Authors: Wu Jing , Thabet Salim , Chen Wei , Goldstein Anna , Madhur Meena , Hudson Billy , Harrison David G
Published: March 2022

Clinical studies show an association between inflammation and arterial stiffness. We and others have shown that T cells are involved in hypertension; however their role in aortic stiffening is unknown. We hypothesized that T cells mediate angiotensin II-induced aortic stiffening. Using a pressurized myograph system, we determined aortic stiffness and quantified this using a distensibility index (DI), defined as the percent change in aortic outer diameter from 75 mmHg to 125 mmHg. Angiotensin II (490ng/kg/min for 14 days) markedly decreased the distensibility index in C57Bl/6 mice (23.8±5.5 v.s. 8.9±4.1, p<0.01, n=8), and this was almost completely prevented in RAG-1
-/-
mice (18.3±4.0, p<0.01 compared with C57Bl/6, n=8). Adoptive transfer of pan T cells into RAG-1
-/-
mice restored aortic stiffness in RAG-1
-/-
(11.9±2.6, p<0.05, n=5). The effect of T cell-derived pro-inflammatory cytokines on aortic stiffening was examined using interferon-γ (IFN-γ
-/-
) and IL-6
-/-
mice. Deficiency of INF-γ partially restored aortic distensibility (17.0±3.5 v.s. 8.9±4.1, p<0.01, n=8) compared C57Bl/6 mice treated with angiotensin II. Aortic distensibility was also preserved in IL-6
-/-
mice (19.9±5.9 v.s. 8.9±4.1, p<0.01, n=8) treated with angiotensin II. In contrast to these functional alterations, the increase in collagen caused by angiotensin II was similar between wild-type and RAG-1
-/-
mice. There was no change in elastin abundance with angiotensin II treatment or in different mouse strains. In conclusion, T cells are essential for angiotensin II-induced collagen deposition, vascular hypertrophy and aortic stiffening. Part of these effects could be mediated by pro-inflammatory cytokines such as INF-γ and IL-6. These likely mediate aortic stiffening by altering properties of elastin and collagen such as crosslinking, rather than alter total abundance of these proteins.
Abstract 163: The Role of Genetic Variants in Arhgef11 via the Rho-rock Pathway on Kidney Injury in the Dahl S Rat
Authors: Jia Zhen , Guo Zibiao , Johnson Ashley C , Garrett Michael R
Published: March 2022

Hypertension plays an important role in the development of chronic kidney disease. The Dahl salt-sensitive (S) rat is a widely studied model of salt-sensitive hypertension that develops concurrent kidney injury and progressive decline in kidney function. Genetic studies using the S rat, along with the kidney injury resistant, but hypertensive SHR (spontaneously hypertensive rat) have identified many genomic regions associated with kidney injury. Through positional cloning approaches,
Arhgef11
, a Rho guanine nucleotide exchange factor has been identified as a strong candidate gene involved in the kidney injury. The S rat exhibits increased expression of
Arhgef11
, up-regulation of Rho-ROCK pathway, and coding sequence variants that may alter protein function compared to the SHR. Inhibition of the Rho-ROCK pathway (using fasudil) in the S resulted in an attenuation of proteinuria, providing evidence the pathway could influence kidney injury. To better understand the role of
Arhgef11
in the Rho-ROCK pathway, genetic knockdown in cell culture was achieved in HEK293 and NRK cell lines using lentiviral shRNA-
Arhfgef11
constructs. Transduced cells achieved ∼70% knockdown of
Arhgef11
protein. RhoA activity was significantly decreased ∼40% (p<0.01) in both transduced cell lines compared to scramble control. Gene expression and protein levels of
Arhgef11
, RhoA, ROCK1, MLC, Cofilin were all significantly down-regulated 40-65% in transduced HEK293 and NRK (p<0.01). Reduced expression of
Arhgef11
in transduced cells showed a different cellular distribution and less stress fiber formation and elongation compared to control (2-fold reduction in length/width ratio, p<0.01) by immunofluorescence. Genetic knockdown demonstrated a similar effect as fasudil for both stable cell-lines. Microarray studies have also identified other novel pathways influenced by genetic knockdown of
Arhgef11
. Current studies are investigating the specific effect of the S or SHR
Arhgef11
allele (using lentiviral overexpression) on the Rho-ROCK pathway and stress fiber formation. In summary, our promising data suggests that the biological and functional significance of genetic variants in
Arhgef11
could be of major importance in development of kidney injury in the S rat.
Abstract 167: Mapping of Chromosome 2 Regions Linked to Vascular Inflammation Using Congenic Rats
Authors: Rehman Asia , Yamamoto Naoki , Mian Muhammad O , Barhoumi Tlili , Kwitek Anne E , Paradis Pierre , Schiffrin Ernesto L
Published: March 2022

Background:
Immune cells have been implicated in hypertension and vascular inflammation. We demonstrated that chromosome 2 modulates immune responses in genetic hypertension via T regulatory lymphocytes (Treg). Introgression of chromosome 2 from normotensive Brown Norway (BN) rats into hypertensive Dahl salt sensitive (SS) background (consomic SB2) reduced vascular inflammation and restored Treg function. We hypothesized that the BN chromosome 2 contains genes that reduce vascular inflammation, which could be mapped using congenic rats containing portions of BN chromosome 2 on the SS background.

Methods:
Twelve-to-13 week old male BN, SS, SB2, congenic (SB)A, SBB and SBE rats fed normal salt diet were studied. Systolic blood pressure (SBP) was measured by telemetry. Spleen Treg (CD4
+
CD25
hi
) and CD4
+
CD25
-
T lymphocytes were isolated and characterized by fluorescence-activated cell sorting (FACS) and cultured. Transforming growth factor (TGF)-β, tumor necrosis factor (TNF)-α, interleukin (IL)-10, IL-17 and IL-6 secreted by 10
5
cells in culture media was measured by microbead multiplex immunoassays. Aortic collagen content was determined by Sirius red staining.

Results:
SS, SB2 and SBE exhibited 20 mmHg higher SBP compared to BN, SBA, and SBB (
P
<0.05). The % of CD4
+
CD25
-
was higher in SS, SB2 and SBE (∼16 %) compared to BN, SBA and SBB (∼12%,
P
<0.05). The % of Treg was lower in SBA (2%) compared to SS and SB2 (3%,
P
<0.05). CD4
+
CD25
-
secretion of TNF-α, IFN-γ and IL-6 was always lower in SS and SBB (≤141, 1428 and 13 pg/10
5
cells, respectively,
P
<0.05), and consistently unchanged in SB2 and SBE (∼320, 22350 and 50 pg/10
5
cells, respectively) compared to BN (458, 3552 and 57 pg/10
5
cells, respectively). Treg IL-10 and IL-17 production was increased in SB2 (9597 pg/10
5
cells), and SS and SB2 (>90 pg/10
5
cells), respectively (
P
<0.05) and unchanged in congenic rats (∼2540 and 23 pg/10
5
cells, respectively), compared to BN (2497 and 9 pg/10
5
cells). Aortic collagen was increased 3-fold in SS, 1.7-fold in SB2 and SBB (
P
<0.05) and unchanged in SBA and SBE compared to BN.

Conclusion:
These results suggest that some of the genes that regulate vascular inflammatory responses are contained within the fragment of chromosome 2 from Brown-Norway rats present in congenic SBE rats.
Abstract 168: Abnormal Pro-inflammatory Regulation of the Innate Immune System in Genetic Hypertension
Authors: Harwani Sailesh , Chapleau Mark W , Legge Kevin , Ballas Zuhair , Abboud Francois M
Published: March 2022

Immune cells from patients with essential hypertension are hyper-responsive to activation of toll-like receptor (TLR) 4 with lipopolysaccharide. Activation of the alpha7 nicotinic cholinergic receptor exerts an anti-inflammatory effect on innate immune cells. We hypothesized that this anti-inflammatory cholinergic response is lacking in the Spontaneously Hypertensive Rat (SHR) and recently reported that nicotine suppresses TLR7 and TLR9 mediated IL-6 secretion by isolated splenocytes of WKY rats; whereas, it dramatically enhances IL-6 secretion in SHR. Here we aimed to confirm these effects in vivo and identify potential cellular mediators of the observed immune responses. Flow cytometry on splenocytes revealed a greater number of CD3-/CD8dim innate immune cells in SHR (4.0±0.56%), compared to WKY (2.97±0.6%)(n=3, p<0.05). Using CD161, a cell surface marker of natural kille/dendritic cells and activated macrophages, we show that the CD3-/CD8dim+/CD161+ and CD3-/ CD8-/CD161+ innate immune cells are increased in SHR (5±0.5% and 4.6±0.85, respectively), compared to WKY (0.8±0.3% and 1.0±0.5%, respectively) (n=3, p<0.05). Moreover, nicotine expanded both cell populations in the SHR to 6.4% and 6.0%, respectively, but had no effect in WKY, where the populations remained at 1.1% and 1.2%, respectively.
In vivo,
we infused saline or nicotine (15mg/kg/day) over 24 hours using subcutaneous pumps. At 20 hrs, animals received an intraperitoneal injection of a TLR7 ligand (Clo97). Serum was collected at the end of the 24 hr infusion. Similar to previous in vitro results, nicotine suppressed the serum IL-6 response to i.p. Clo97 from 108±28 pg/ml in WKY (n=4) to 12±7 pg/ml (p<0.05) in 4-5 week old WKY, but dramatically enhanced the IL-6 response in SHR (n=4), following i.p. Clo97, from 58±20 pg/ml to 187±38 pg/ml (p<0.05). We conclude that there is a dominant innate immune cell population in SHR prior to the development of hypertension that expands with cholinergic stimulation and may contribute to the pronounced pro-inflammatory increase in IL-6 following cholinergic stimulation. We speculate that this abnormality in the pre-hypertensive state contributes to the development of genetic hypertension and may play a role in human essential hypertension.
Abstract 17: Prolonged ER Stress Induced by Angiotensin II-Hypertension Contributes to Aortic Stiffening via an Increase in Pro-apoptotic and Pro-inflammatory Signaling Pathways
Authors: Spitler Kathryn M , Webb R. C
Published: March 2022

Aortic stiffening is an important predictor of negative cardiovascular outcomes and is associated with hypertension. Angiotensin II (Ang II) is a mediator of aortic stiffening via upregulation of pro-inflammatory and pro-apoptotic signaling pathways, however the mechanisms mediating this upregulation are unclear. Endoplasmic reticulum (ER) stress occurs during cardiovascular diseases, leads to upregulation of pro-inflammatory and pro-apoptotic pathways and can be induced by Ang II. We hypothesized that ER stress leads to vascular apoptosis and inflammation contributing to increased aortic stiffening during Ang II-induced hypertension. Twelve-week old Sprague-Dawley rats were implanted with mini-osmotic pumps containing Ang II (60ng/min/day, 28 days) or received sham surgery and co-treated with ER stress inhibitors, tauroursodeoxycholic acid (TUDCA) or 4-phenylbutyric acid (PBA) (100mg/kg/day, i.p. 28 days) or saline. Thoracic aorta was used for functional myograph analysis and western blot (data presented as fold change from control ± SEM). Aortic rings from the Ang II saline group had a greater contraction to phenylephrine compared to control (Emax: 33.1±0.7 mN vs 26.8±0.6 mN) which was attenuated by ER stress inhibition (Emax: 23.4±0.4 mN,PBA; 26.05±0.7 mN,TUDCA). ER stress inhibition suppressed NF-κB phosphorylation (1.0±0.4, PBA; 1.3±0.2, TUDCA) compared to Ang II saline group (1.9± 0.4). ER stress inhibition reduced pro-apoptotic protein Bax expression (1.3± 0.7, PBA; 1.8± 0.1, TUDCA) compared to Ang II saline group (2.5±0.7) and increased anti-apoptotic protein Bcl-2 expression (1.2±0.5, PBA; 1.3±0.4, TUDCA) compared to Ang II saline group (0.6 ±0.2). Collagen type I and type III expression was attenuated with ER stress inhibition (1.5±0.2, PBA; 1.4±0.4, TUDCA and 1.0±0.3, PBA; 0.9±0.4 TUDCA, respectively) compared to Ang II saline group (3.7±0.4 and 1.8±0.3, respectively). These effects were independent of blood pressure changes as ER stress inhibition did not alter systolic blood pressure (163±3 mmHg, PBA; 170±4 mmHg, TUDCA) compared to Ang II saline group (165±4 mmHg). Our data suggest that ER stress is a new mechanism through which Ang II promotes vascular inflammation and apoptosis resulting in increased aortic stiffness.
Abstract 170: Aldosterone Activates ex vivo Human Polymorphonuclear Leukocytes
Authors: Maldonado Enrique R , Prado Gregory N , Nieva Adriana , Vazquez Carlos , Saca Gabriel , Rivera Alicia , Romero Jose R
Published: March 2022

An important role for the mineralocorticoid receptor (MR) in vascular inflammation has been proposed. Polymorphonuclear (PMN) leukocytes are critical players following an inflammatory response. We studied the role of aldosterone (ALDO) on ex vivo PMN function by isolation of cells from circulating human blood following density gradient sedimentation with polymorphprep from otherwise healthy subjects. These cells express MR as confirmed by western blot, qRT-PCR and 3H-aldosterone binding analyses. Incubation of ex vivo PMN cells with ALDO (1-10 nM) showed a rise in superoxide production (P<0.01, n=9), an event that was blunted by pre-incubation with 500 nM canrenoic acid (CA), a MR antagonist (P<0.03, n=6). Consistent with these results, we observed that 10 nM ALDO led to rapid increases in cytosolic Ca2+ levels using FURA-2AM fluorescence and was associated with decreases in cellular Mg2+ levels using MagFURA-2AM following 1.5 hr of ALDO. These events were associated with increases in VEGF secretion from PMN as determined by ELISA of supernatants (260.64±44.6 vs. 134.03±1.99 pg/ml, P<0.01 when compared to vehicle, n=8) that was likewise sensitive to CA (P<0.04, n=8); thus suggesting that ALDO-activated PMN cells increase factors involved in migration and endothelial cell activation. Indeed, we showed that 10 nM ALDO increased PMN cell migration in a time-dependent manner by CyQuant fluorescence that lasted up to 60 min and could be blocked by CA (P<0.01, n=3). We then studied the effect of 10 nM ALDO-stimulated PMN cells followed by co-incubation of these cells with human endothelial cells for 4 hr. We observed that incubation of ALDO-stimulated PMNs vs vehicle treated cells led to a 2.1 fold increase in ICAM-1 expression levels using qRT-PCR but not VCAM-1 (P<0.01, n=3) in endothelial cells. We observed similar findings in DMSO-differentiated HL-60 cells, a neutrophil-like human cell line. We detected the presence of MR by western blot and qRT-PCR in these cells. Incubation with 10 nM ALDO led to increases in cellular calcium, superoxide production (P<0.01, n=3) and HL-60 migration (P<0.03, n=4) when compared to vehicle. Thus our results suggest that activation of MR leads to PMN cell activation that may contribute to vascular inflammatory responses in vivo.
Abstract 173: Depletion of CLIP+ Immune Cells Prevents Toll-Like Receptor-Induced Preeclampsia in Mice
Authors: Seerangan Geetha , de Guzman Eugene V , Chiasson Valorie L , Chatterjee Piyali , Tobin Richard P , Young Kristina J , Newell M. K , Mitchell Brett M
Published: March 2022

Preeclampsia (PE) is a pregnancy-specific disorder of vascular endothelial dysfunction and inflammation which manifests as hypertension and proteinuria and may result from maladaptation of the maternal immune system. Activation of the dsRNA receptor Toll-like receptor 3 (TLR3) or the ssRNA receptor TLR7 elicits pregnancy-dependent hypertension and endothelial dysfunction in mice in part by increasing pro-inflammatory immune cells. Polyclonal activation of immune cells is associated with expression of Class II-Associated Invariant Peptide Chain (CLIP) in MHC class II which prevents T cell-dependent death leading to persistent immune cell activation. We hypothesized that removal of CLIP reduces pro-inflammatory immune cells and prevents the development of PE in mice. Pregnant mice were given ip injections of normal saline (P), poly I:C (TLR3 agonist; P-PIC), or R837 (TLR7 agonist; P-R837) with or without TPP, a custom-targeted peptide that removes CLIP from MHC class II, on gestational days 13, 15, and 17 and euthanized on day 18. TPP treatment significantly decreased pro-inflammatory CD3+/γδ T cells and increased anti-inflammatory CD4+/CD25+ regulatory T cells in TLR3-induced PE mice as well as TLR7-induced PE mice. TPP treatment also prevented the hypertension (GD17 SBP in mmHg: P=102±3, P+TPP=105±4, P-PIC=147±4*, P-PIC+TPP=103±2, P-R837=133±2*, P-R837+TPP=106±4; *p<0.05 vs. P), proteinuria, and endothelial dysfunction in TLR3-induced and TLR7-induced PE mice while having no detrimental effects in normal pregnant mice. Based on our results, the TPP-mediated decrease in detrimental immune cells positively modulated T cell subsets and prevented the development of PE-like symptoms in mice. CLIP removal from MHC class II with TPP may be a promising therapy for women with PE.
Abstract 174: T Cells Promote the Progression of Hypertension and Renal Injury during Systemic Lupus Erythematosus
Authors: Mathis Keisa W , Wallace Kedra , Wasson Katie L , Masterson C. W , LaMarca Babbette , Ryan Michael J
Published: March 2022

Systemic lupus erythematosus (SLE) is an autoimmune disorder with prevalent hypertension and is a novel disease model to study the link between immune system activation and the development of hypertension. T cells have a central role in the pathogenesis of autoimmune disorders and were recently reported to promote angiotensin II and salt-sensitive hypertension. Despite their well known role in the pathogenesis of autoimmune disease and recent data suggesting an important role in the pathogenesis of hypertension, whether T cells mechanistically contribute to the prevalent hypertension during SLE remains unclear. In order to test this, experiments were designed to determine whether inhibition of T cell proliferation attenuates the progression of hypertension and renal injury in female SLE mice with established renal injury (albuminuria ≥ 100 mg/dL). SLE and control mice were administered either antibody to CD3, a component of the T cell receptor expressed on all T cells, or an anti-IgG isotype control for up to 4 weeks (intranasally; 25 μg/week). Splenic CD3
+
CD4
+
T cells were increased in SLE mice compared to controls (7.0 ± 4.2% vs. 3.1 ± 1.5% gated) and were reduced after the anti-CD3 treatment (2.4 ± 1.1% gated), likely reflecting a reduction in spleen weight. Anti-dsDNA autoantibodies characteristic to SLE were increased in vehicle-treated SLE mice compared to controls (19.8 ± 7.3 vs. 0.74 ± 0.07, n=10) and were blunted in the anti-CD3 treated SLE mice (9.7± 3.8, n=10). Blood pressure (mmHg) was increased in SLE mice compared to controls (156 ± 3 vs. 112 ± 4, p=0.001), and was lower in anti-CD3 treated SLE mice (137 ± 4; p=0.01) but not controls (117 ± 2). In anti-IgG treated SLE mice, albuminuria increased (+38.9 ± 36.8%) over the course of the study, whereas albuminuria in anti-CD3 treated SLE mice was unchanged (-2.53 ± 17.3%). Preliminary renal histology suggests that the anti-CD3 treatment attenuated the progression of glomerulosclerosis and preserved renal vascular structure (i.e., interlobular arteries, vasa recta). No renal injury was observed in the control mice. These data suggest that T cells have an important mechanistic role in the progression of autoimmune-associated hypertension, perhaps by promoting renal vascular structural changes and injury.
Abstract 181: Toll-Like Receptor 9 Activation Leads to Augmented Thromboxane A2 Induced Contractions in Rat Aorta
Authors: McCarthy Cameron G , Goulopoulou Styliani , Matsumoto Takayuki , Webb R. Clinton
Published: March 2022

Evidence has supported the involvement of the immune system in the development of vascular diseases. Toll-like receptor 9 (TLR 9) is a pattern recognition receptor of the immune system which is activated by exogenous DNA (bacterial and viral) and endogenous mitochondrial DNA (mtDNA) containing unmethylated CpG motifs. Activation of TLR 9 in immune cells leads to the release of pro-inflammatory cytokines via nuclear factor kappa B (NF-κB) and mitogen-activated protein kinase (MAPK) signaling. TLR 9 is also expressed in the vasculature; however, its role in vascular diseases remains to be elucidated. Therefore, we hypothesized that TLR 9 activation, using a synthetic oligonucleotide containing unmethylated CpG motifs [ODN 2395 (ODN); 10
-5
M], would augment aortic contractility in female, 16 weeks old, Sprague Dawley rats. Contractile concentration response curves were performed
ex vivo
using the myograph to thromboxane A
2
(TxA
2
) mimetic (U-46619; 10
-9
-3x10
-6
M) and phenylephrine (PE; 10
-9
-3x10
-5
M), following incubation for 15 hours with ODN or vehicle (Veh). Results revealed that ODN amplified the contractile responses to U-46619 [E
max
(% max KCl), ODN: 262±18 vs. Veh: 178±13; Fig. A]. Moreover, ODN attenuated aortic sensitivity to PE compared to Veh [pD
2
, ODN: 6.4±0.1 vs. Veh: 6.8±0.2; Fig. B]. These data suggest that TLR 9 activation heightened aortic contractility to U-46619. We propose that mtDNA activation of TLR 9, released during necrotic events of vascular remodeling, has pro-inflammatory effects that contribute to increased TxA
2
receptor-associated contractions. This could potentially lead to vascular dysfunction and vascular diseases such as hypertension.
Abstract 182: Reduction in Interleukin-10 Level in Plasma and in Renal Tissue During Systemic Inhibition of Nitric Oxide Synthase in Anesthetized Mice
Authors: Singh Purnima , Castillo Alexander , Johnson Tanya R , Dewan Majid S
Published: March 2022

Systemic inhibition of nitric oxide synthase (NOS) in mice increased tumor necrosis factor-alpha (TNFα) level in plasma. The present study examined the hypothesis that an inhibition of interleukin-10 (IL-10; anti-inflammatory cytokine) in response to NOS inhibition facilitates such enhancement in TNFα production. Levels of IL-10 and TNFα were measured (using ELISA kits) in plasma and in renal tissues obtained from anesthetized mice (C57BL6; ∼10 wks age) infused with or without NOS inhibitor, nitro-L-arginine methyl ester (L-NAME; 200 μg/min/kg, iv). Protein expressions of IL-10 and TNFα in the renal tissues were also measured by immuno-staining. L-NAME infusion alone for 130 min (n=6) resulted in decreased IL-10 levels in plasma (0.3±0.1 vs 2.6±0.6 ng/mL) and in renal tissue (0.5±0.1 vs 1.3±0.1 ng/mg protein) which were associated with increased TNFα levels in plasma (432±82 pg/mL vs undetected) and in renal tissue (58±7 vs 6±5 pg/mg protein) as compared to vehicle treated control mice (n=6). There was marked down-regulation of IL-10 protein expression (present only in the distal tubules in control mice) and up-regulation of TNFα protein expression (distal tubules and collecting ducts) in the renal tissues in L-NAME treated mice. Infusion of NO donor, S-nitroso-N-acetyl-penicillamine (SNAP; 25 μg/min/kg for 75 min) in a separate group of L-NAME treated mice (n=6) restored the IL-10 level in plasma (1.0±0.1 ng/mL) and in renal tissue (1.5±0.1 ng/mg protein) closer to the values in control mice indicating that L-NAME induced IL-10 responses are specific to NO inhibition. SNAP infusion in L-NAME treated mice also reduced TNFα level in plasma (to an undetected level) and in renal tissue (20±4 pg/mg protein) along with its protein immune-expression. Infusion of mouse IL-10 (0.075 ng/min/g for 75 min) in another separate group of L-NAME treated mice (n=7) also attenuated TNFα level in plasma (50±16 pg/mL) and in renal tissue (21±7 pg/mg protein) along with its protein immune-expression indicating that L-NAME induced TNFα responses are specific to reduction in IL-10 level. The results demonstrate that systemic NOS inhibition decreases IL-10 production and thus, minimizes its immune down-regulating action on the production of TNFα, particularly in the kidney.
Abstract 188: Yogurt Consumption, Blood Pressure, and Incident Hypertension: A Longitudinal Study in the Framingham Heart Study
Authors: Wang Huifen , Livingston Kara A , Meigs James B , Jacques Paul F
Published: March 2022

Background
Yogurt is a nutrient-dense, low-fat dairy product. We aimed to examine the longitudinal association of yogurt consumption with blood pressure (BP) levels and hypertension prevention among adults participating in the Framingham Heart Study Offspring Cohort (exams 5 to 8 [1998-2001 to 2005-2008]).

Methods
In each exam, dietary intake was assessed by a validated food frequency questionnaire; BP was measured following standardized procedures; and anti-hypertensive medication use was obtained by physician-elicited self-report. Our analyses included 2,197 adults (with 7351 observations) who were free of hypertension at exam 5 (baseline) and had valid measurements of yogurt intake and BP. Logistic regression was used to examine incident hypertension (defined as systolic BP (SBP) ≥140mmHg, or diastolic BP (DBP) ≥90mmHg, or used anti-hypertensive medication). A repeated measures model was used to examine annualized BP change in exam intervals adjusting for BP and anti-hypertensive medication use at the beginning of each interval. All analyses were adjusted for demographic and lifestyle factors (including diet quality).

Results
At baseline, 44% (960 of 2197) of participants consumed ≥1 serving of yogurt per month (i.e. defined as consumers); and mean [SD] SBP and DBP were 117 [12] mmHg and 72 [8] mmHg, respectively. BP and yogurt intake both increased over the 14-year follow up; and 913 participants developed incident hypertension. After adjustment for demographic and lifestyle factors and cholesterol-lowering medication use, people who consumed >2% of total calories (kcal) from yogurt (i.e. high-intake group) had lower risk of incident hypertension than non-consumers (OR [95%CI] = 0.69 [0.54-0.87]). The high-intake group had 0.19±0.09 mmHg smaller annualized elevation of SBP than non-consumers (
P
=0.04). Excluding the anti-hypertensive medication users at follow-up strengthened the longitudinal association between yogurt intake and annualized SBP change. All findings retained when further controlling for body mass index (BMI) and the change of BMI. Yogurt intake was not related to DBP change over time.

Conclusion
Higher yogurt intake, as part of a healthy diet pattern, may be beneficial for BP control and hypertension prevention.
Abstract 194: Angiotensin Ii Induces Fat1 Overexpression and Vascular Smooth Muscle Cell Migration Via Nox1-dependent Reactive Oxygen Species Generation.
Authors: Bruder-Nascimento Thiago , Chinnasamy Prameladevi , Riascos-Bernal Dario , Tostes Rita de Cassia , Sibinga Nicholas E
Published: March 2022

Fat1 is an atypical cadherin that has been implicated in the control of vascular smooth muscle cell (VSMC) proliferation and migration. NADPH oxidase 1 (Nox1) belongs to the NADPH oxidase family and is an important source of reactive oxygen species (ROS) in VSMCs. Angiotensin II (AngII) induces the expression and/or activation of both Fat1 and Nox1 proteins. We tested the hypothesis that AngII-induced Fat1 activation and VSMC migration are regulated by Nox1-dependent ROS generation. Cultured VSMCs from aortae of adult Sprague-Dawley rats were used. Cells were stimulated for 12h with AngII (1 μmol/L), in the presence or absence of Tempol (1 μmol/L), Apocynin (10 μmol/L), or Valsartan (1 μmol/L). ROS were measured by dihydroethidium and lucigenin assays. Nox1 and/or Fat1 expression was knocked down by siRNA and assessed by western blot analysis with quantitation by densitometry. Cellular migration was evaluated using a Transwell system. AngII stimulated ROS in VSMCs as expected. This effect was blocked by siRNA to Nox1 and by apocynin or valsartan. AngII increased Fat1 (2.17±0.24 arbitrary units (au) vs Ctrl 1.02±0.10, p<0.05), and Nox1 expression (1.95±0.15 au vs Ctrl 0.86±0.15, p<0.05). Tempol, apocynin, valsartan, and Nox1 siRNA prevented AngII-induced Fat1 expression (p<0.05), whereas Fat1 siRNA did not change Nox1 expression. AngII increased VSMC migration (440.7±83.4 cells migrated vs Ctrl 189.0±15.72, p<0.05), and this increase was inhibited by apocynin and valsartan (p<0.05), as well as by Nox1 (227.0±44.27) and Fat1 (192.3±67.4) siRNA (p<0.05). In conclusion, our findings indicate that AngII enhances Fat1 expression and Fat1-dependent VSMC migration via AT1 receptor- and Nox1-dependent ROS generation, and link AngII effects on vascular remodeling to regulation of Fat1 expression.
Abstract 203: Transcriptomic Analysis of Placental Chorionic Villi During Placental Ischemia-induced Hypertension
Authors: George Eric , Granger Joey P
Published: March 2022

While the precise etiology of preeclampsia is unclear, it is believed that a central causative agent of the disorder is placental ischemia caused by placental hypoperfusion and chronic ischemia. This results in the secretion of pathogenic factors into the maternal circulation. While a number of these factors have been identified (i.e. sFlt-1, AT1-AA, inflammatory cytokines), the full pathogenic profile remains unresolved. Here we have attempted to identify novel biomarkers and pathogenic factors in the ischemic placenta by quantitative microarray analysis of both mRNA and miRNA expression in a rodent model of chronic placental ischemia, the Reduced Uterine Perfusion Pressure (RUPP) model. Gene expression arrays from chorionic villi from control and RUPP animals (n=6 each) revealed 2557 differentially expressed genes at a statistical cutoff of p<0.05 (1140 up-regulated, 1417 down-regulated. The thirty most differentially affected transcripts had fold changes of 2.6-1.4 and included several genes previously identified in the placenta of human patients and experimental models, including pappalysin-2, CEACAM1, and heme oxygenase-1. Most pronounced was a significant increase in the expression of 9 prolactin family members, indicating a massive upregulation of the prolactin system. In addition, a number of novel pro-inflammatory and anti-angiogenic targets were identified. The expression of miRNA in these samples was also determined, and at a statistical cutoff of p<0.05 there were 577 down-regulated and 458 up-regulated miRNAs, of which 73 were changed by more than 50%. Among the pathways known to be affected by these miRNA’s are the VEGF signaling pathways, HMOX1, the Notch pathway, and the HIF activator p300, all of which have previously been implicated in the etiology of preeclampsia. Functional analysis of these transcripts could provide important mechanistic insight into the regulatory mechanisms behind the development of preeclampsia. Together, these data provide interesting biomarkers for placental ischemia and could suggest new therapeutic targets for the treatment of preeclampsia.
Abstract 209: Hemeoxygenase-1 Induction Restores Beta ENaC Expression in Ischemic Placentas
Authors: Warrington Junie P , George Eric M , Ryan Michael J , Drummond Heather A , Granger Joey P
Published: March 2022

Preeclampsia is a pregnancy-specific disease characterized by hypertension, proteinuria, and vascular abnormalities; the etiology of which is under active investigation. Utilizing the rat model of reduced uterine perfusion pressure (RUPP), our lab previously demonstrated that induction of hemeoxygenase-1 (HO-1) reduces blood pressure and oxidative stress in placental ischemic rats. While epithelial sodium channel proteins (specifically β-ENaC) are important in maintaining vascular myogenic tone and have been implicated in cytotropholast invasion, the effect of placental ischemia and induction of HO-1 on β-ENaC expression in placentas have not been established. Therefore, the purpose of this study is to test the hypothesis that placental β-ENaC expression is reduced in ischemic placentas and restored by HO-1 induction. Normal pregnant and RUPP rats were treated with the HO-1 inducer, cobalt protoporphyrin (CoPP) (n=4 per group/treatment). Blood pressure was measured and placentas were harvested on day 19 of gestation. Blood pressure increased from 101±1 to 141±4 mmHg (p<0.0001) in response to the RUPP procedure but was reduced by HO-1 induction (117±2 mmHg). RUPP rats also had fewer pups compared to normal pregnant rats (14±1 vs. 4±2 pups). HO-1 induction did not prevent the reduction in pup number. Placental β-ENaC protein expression was reduced by 62% in the RUPPs compared to the normal pregnant rats (p=0.036). HO-1 induction restored placental expression of β-ENaC to the levels of the normal pregnant rats. These findings suggest that HO-1 may exert its protective effects by altering the placental environment through changes in β-ENaC expression.