bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 69: Role of Neutrophil Gelatinase Associated Lipocalin in Cardiovascular Remodeling Induced by Aldosterone
Authors: Tarjus Antoine , El Moghrabi Soumaya , Latouche Céline , Rossignol Patrick , Zannad Faiez , Farman Nicolette , López-Andrés Natalia , Jaisser Frédéric
Published: March 2022

Neutrophil Gelatinase Associated Lipocalin (NGAL) is a circulating protein, member of the lipocalin family, which binds MMP9 and modulates its stability and activity. We have recently shown that NGAL is a primary target of aldosterone/mineralocorticoid receptor (MR) in endothelial cells, vascular smooth muscle cells and cardiomyocytes. We hypothesized that NGAL could be a mediator of aldosterone/MR profibrotic and proinflammatory effects in the cardiovascular system. Wild type (WT) and NGAL Knock Out (KO) mice were subjected to an uni-nephrectomy aldosterone salt challenge (NAS, 200μg/kg/day of aldo, 1% NaCl in tap water) for 4 weeks. Blood pressure (SBP) was measured by tail cuff method. Vascular reactivity was assessed by wire myograph. Cardiovascular fibrosis and inflammation were analyzed by RT-PCR, western blot, immunohistochemistry and ELISA. There was no difference in SBP between NGAL KO mice compared to WT mice in basal condition. With NAS challenge, SBP was increased only in WT (SBP; CT 107±3, CT NAS 133±5, KO 109±3, KO NAS 115±3 mmHg). Ex vivo aortic contraction induced by phenylephrine (Phe) and potassium chloride (KCl) were increased by NAS challenge in WT and KO (Emax Phe; CT 2.28±0.8, CT NAS 10.65±1.3, KO 1.45±0.3, KO NAS 9.32±0.9 mN). Responses to vasodilators (acetylcholine and sodium nitroprussiate) were altered in NAS condition in both groups. Quantification of pro collagen I N-terminal peptide (PINP) in plasma showed an increase of PINP due to NAS treatment in WT that was prevented by NGAL inactivation (CT 83±15, CT NAS 129±10, KO 70±19, KO NAS 59±12 μg/l). In myocardium, NAS treatment increased collagen type I and perivascular fibrosis in WT whereas KO were resistant to fibrosis (Collagen Volume Fraction; CT 19±3, CT NAS 28±2, KO 20±3, KO NAS 20±3 %). In aorta, collagen type I, vascular fibrosis and osteopontin were also increased by NAS in WT. These increases were prevented by NGAL inactivation (CVF; CT 24±4, CT NAS 34±5, KO 21±2, KO NAS 28±2 %). Our results show that NGAL plays a role in aldosterone/MR-mediated hypertension, vascular and cardiac peri-vascular fibrosis and inflammation, but not in vascular dysfunction.
Abstract 7: Both Maternal Systemic and Paternal-Derived Placental Trophoblast TLR3 Activation Are Necessary for the Development of Preeclampsia in Mice
Authors: Chatterjee Piyali , Chiasson Valorie L , Kopriva Shelley E , Mitchell Brett M
Published: March 2022

Preeclampsia (PE) is a pregnancy-specific hypertensive disorder which affects ~8% of all pregnancies worldwide. The placenta is considered to be the main culprit in the etiology of PE as the symptoms of PE typically abate following removal of the placenta. PE is also known to be mediated in part by excessive maternal immune system activation and inflammation. We developed a mouse model of PE by excessively activating TLR3, which recognizes dsRNA from necrotic tissue and viruses; however, it is unknown whether TLR3 activation in maternal systemic cells, in paternally-derived placental trophoblasts, or both play a role in the development of hypertension during pregnancy. We hypothesized that both contribute and to test this we used the following mating schemes: 1) female WT mice mated with male WT mice (control), 2) female TLR3 KO mated with WT male (Ma TLR3 KO, TLR3 deficiency in all maternal cells), 3) male TLR3 KO mated with female WT (Pa TLR3 KO, TLR3 deficiency in paternally-derived trophoblasts, the major cell type in the placenta), and 4) female TLR3 KO mated with male TLR3 KO (MaPa TLR3 KO, TLR3 deficiency in all maternal cells and trophoblasts). Pregnant WT and TLR3 KO (Ma, Pa, and MaPa) mice were subjected to poly I:C or saline treatment on gestational days 13, 15, and 17. Poly I:C treatment induced pregnancy-dependent hypertension in pregnant WT mice (PPIC WT) (day 17 SBP in mmHg: PPIC WT: 147±5 vs. P WT: 100±4; p<0.05), however this was attenuated in PPIC Ma TLR3 KO mice (109±2 mmHg; p<0.05 vs. P WT) and PPIC Pa TLR3 KO mice (109±1 mmHg; p<0.05 vs. P WT), and completely normalized in PPIC MaPa TLR3 KO mice (102±3 mmHg; P>0.05 vs. P WT). PPIC WT mice exhibited endothelial dysfunction, proteinuria, and increased fetal demise compared to P WT mice which were attenuated in PPIC Ma TLR3 KO and PPIC Pa TLR3 KO mice, but completely normalized in PPIC MaPa TLR3 KO mice. PPIC WT mice exhibited splenomegaly which was only completely reduced in PPIC MaPa TLR3 KO mice. In conclusion, our data suggest that both maternal systemic and paternal-derived placental trophoblast TLR3 activation contribute to the full development of PE-like symptoms in mice.
Abstract 8: Delivery of an Anti-inflammatory NF-κB Inhibitory Polypeptide to Treat Preeclampsia
Authors: George Eric M , Liu Huiling , Bidwell Gene L
Published: March 2022

Preeclampsia is a hypertensive disorder of pregnancy that is a leading cause of maternal, fetal, and perinatal morbidity and mortality. There is currently no effective intervention for preeclampsia short of induced delivery of the fetus. Improvements in preeclampsia management have been largely stifled due to deleterious effects of proposed small-molecule drugs on the developing fetus. The progression of preeclampsia is driven by two major pathways, secretion of the VEGF antagonist sFlt-1 and induction of a highly inflammatory environment in the mother. Our lab is developing therapeutics to target each of these pathways. To inhibit the maternal inflammatory response, we have developed an NF-κB inhibitory peptide therapy. This NF-κB inhibitory peptide is attached to a drug delivery vector called elastin-like polypeptide (ELP) that stabilizes it from rapid clearance and degradation in the maternal circulation while preventing it from crossing the placenta into the fetal circulation. When administered to pregnant rats on gestational day 14, ELP accumulated highly in the placenta but was barely detectable in the pups (placenta ELP levels were 17.9 fold higher than pup levels, p = 0.00058). The ELP-delivered NF-κB inhibitory peptide was highly active against the NF-κB pathway in human umbilical vein endothelial cells (HUVECs). TNFα stimulation led to NF-κB activation in HUVECs as evidenced by nuclear localization of the NF-κB p65 subunit (nuclear:cytoplasmic ratio increased from 0.76 to 1.79 after stimulation). This activation was completely blocked by pretreating the cells with the NF-κB inhibitory polypeptide (nuclear:cytoplasmic ratio of ~ 0.9 before and after TNFα stimulation), but not by a control polypeptide. Also, pretreatment of HUVEC cells with the NF-κB inhibitory polypeptide completely blocked TNFα stimulated endothelin-1 release from HUVECs, suggesting that this strategy will be effective for ameliorating pregnancy induced hypertension. The polypeptides had no effect on HUVEC proliferation. These results suggest that the ELP-delivered NF-κB inhibitory peptide is a strong inhibitor of the inflammatory response in vascular endothelial cells, is nontoxic, and can be delivered to the mother while being excluded from the fetus.
Abstract 82: Critical Role of Striatin in Vascular Responses to Sodium Intake
Authors: Garza Amanda E , Pojoga Luminita H , Opsasnick Lauren A , Siddiqui Waleed T , Romero Jose R , Adler Gail K , Williams Gordon H , Khalil Raouf A
Published: March 2022

Striatin is a scaffolding protein that plays a role in vesicular trafficking in neurons. Striatin binds to regulatory proteins such as caveolin-1, Ca2+-calmodulin, Gαi and phosphatase 2A, thereby activating transduction molecules such as eNOS and mitogen-activated protein kinase. We have shown that striatin colocalizes with the minerlacorticoid receptor (MR), and that MR activation increases striatin levels in vascular cells in vivo and in vitro. To test the hypothesis that striatin is a critical regulator of vascular function, WT mice and heterozygous striatin-deficient mice lacking one allele for striatin (Strn-HET) were randomized in a crossover intervention to liberal salt (HS, 1.6% NaCl) and low salt (LS, 0.03% NaCl) diets for 7 days on each diet. Blood pressure (BP) and plasma aldosterone (ALDO) levels were measured and the aorta was excised to assess vascular function. Δ Systolic BP (BP on HS - BP on LS) was in Strn-HET > WT (16.5±3.5 vs 3.7±6.1 mmHg). Plasma ALDO levels were higher during LS diet, but were similar in WT and Strn-HET mice (152.6±23.9 vs 131.5±47.6 ng/dL on LS; 72.9±7.7 vs 70.9±8.3 ng/dL on HS). Phenylephrine (Phe) caused vascular contraction that was in Strn-HET (max 0.31±0.07g) > WT (0.23±0.07g). Also, high KCl (96 mM)-induced contraction was in Strn-HET (0.44±0.06g) > WT mice (0.33±0.05g) on HS diet, suggesting that the enhanced vascular contraction is not limited to a particular receptor. Endothelium removal, NOS inhibitor L-NAME and guanylate cyclase inhibitor ODQ enhanced contraction and increased sensitivity to Phe in WT mice and to a greater extent in Strn-HET mice on HS but not LS diet. On HS, acetylcholine (ACh) relaxation was in Strn-HET (max 33.16±4.47%) < WT (50.94±10.88%). ACh relaxation was blocked by endothelium removal, L-NAME and ODQ, supporting a role of the NO-cGMP pathway. Vascular relaxation to the exogenous NO donor sodium nitroprusside was not different between groups. Thus striatin deficiency during HS diet is associated with salt sensitivity of BP, enhanced vasoconstriction and decreased vascular relaxation. The results suggest a critical role for striatin, possibly through modulation of endothelium-dependent NO-cGMP pathway, in regulation of vascular function and BP during changes in dietary sodium intake.
Abstract 83: Contribution of Nox4 to Luminal Flow Induced H2O2 production in Medullary Thick Ascending Limbs of Dahl Salt-Sensitive Rats
Authors: Zheleznova Nadezhda N , Geurts Aron M , Yang Chun , Cowley Allen W
Published: March 2022

Multiple isoforms of NADPH oxidases (Nox) have been identified in the kidney including Nox1, Nox2, and Nox4 which contribute to elevated levels of renal oxidative stress in hypertension. However, the relative contribution and the mechanisms of their regulation of these specific isoforms remain unclear. The role of Nox4 has received the least attention even though it is the most highly expressed isoform in the kidney. It is also unique in that it is thought to release primarily H
2
O
2.
In previous studies using Sprague Dawley rats, we found that increased luminal flow and Na
+
delivery increased intracellular H
2
O
2
production in epithelial cells of the medullary thick ascending limb of Henle (mTAL). In the present study, we determined whether Nox4 contributed to the flow induced increases of mTAL H
2
O
2
. As part of a broader study, microdissected-isolated mTAL from Dahl salt-sensitive (SS) rats fed a 0.4% NaCl since weaning were examined and the presence of Nox4 mRNA confirmed.
Ex vivo
studies were then performed to determine if absence of Nox4 reduced H
2
O
2
production in response to increases of mTAL luminal perfusion from 5 to 20 nl/min. Isolated microperfused mTAL tubular responses of SS rats fed a 4% NaCl diet for 3 days were compared to SS Nox4 null mutant rats (SS
Nox4-/-
) which were created using zinc finger nuclease techniques. A sensitive intracellular fluorescent probe (PF6-AM), highly specific to H
2
O
2
,

was used to quantify responses to luminal flow. Results showed that H
2
O
2
production following the increase in luminal perfusion rate was reduced in SS
Nox4-/-
rats nearly 85% compared to SS (1300±138 Fluorescent Units (FU); n=5) versus 244±185 FU (n=8; p<0.05). We conclude that Nox4 is of major importance in the production of H
2
O
2
in response to an increase in luminal flow and Na
+
delivery in the mTAL of SS rats and could represent an important therapeutic target for salt-sensitive hypertension.
Abstract 92: Low-intensity Resistance Exercise Training Improves Cardiovascular Dysautonomia and Systolic Function in Diabetic Rats
Authors: Moraes Oscar A , Scapini Kátia B , Arruda Paula , Cardoso Ruymar , Mostarda Cristiano , De Angelis Kátia , Irigoyen Maria C
Published: March 2022

Background:
Cardiovascular complications are the major cause of death in diabetic patients. Diabetic rats by streptozotocin (STZ) administration present many alterations similar to those occurring in humans like diabetic cardiomyopathy and neuropathy, impairing the cardiovascular autonomic control. Aerobic exercise training improves these alterations, however evidence about the effects of resistance exercise on these parameters are scarce.

Objective:
To evaluate the effects of low-intensity resistance training in baroreflex sensitivity (BRS), in autonomic control and cardiac function in STZ diabetic rats.

Methods:
Male Wistar rats (3 months of age) were divided into sedentary control group (SC, n = 8), sedentary diabetic (SD, n = 8), trained diabetic (TD, n = 8) and trained control (TC, n = 8). Trained groups underwent low-intensity resistance training (40-50% maximal voluntary contraction) for 10 weeks. All animals were instrumentalized with femoral artery and vein cannulas. Blood pressure (BP) and heart rate (HR) signals, baroreflex sensitivity (BRS) and autonomic tonus were evaluated, as well as cardiac function by echocardiography.

Statistical analysis:
Data were compared by two-way ANOVA followed by Student Newmann Keuls post hoc test. Significance level was p<0.05.

Results:
Diabetic rats showed lower systolic BP and HR, impairment of BRS and autonomic function and systolic and diastolic dysfunction in comparison with controls. Resistance training did not change glycemia, however it attenuated the resting hypotension (SD: 105±4 vs. TD: 117±4 mmHg) and bradycardia (SD: 283±4 vs. TD: 16±13 bpm), improved the reflex bradycardia (SD: -0.57±0.08 vs. TD: -1.75±0.1 bpm/mmHg) and the reflex tachycardia to BP changes (SD: 1.08±0.2 vs.TD: 3.10±0.15 bpm/mmHg), the vagal tonus (SD: 13±3 vs. TD: 43±4 bpm), as well as the ejection fraction (SD: 68±2 vs. TD: 75±3 %) of diabetic rats.

Conclusion:
Low-intensity resistance training attenuated the hemodynamic impairment, improved BRS, autonomic control and systolic function in STZ diabetic rats without modifying the hyperglycemia. These data suggest that resistance exercise training induced-cardiovascular improvement can be independent of changes in glycemic control in a model of type 1 diabetes.
Abstract 93: In Vivo Two-photon Fluorescence Microscopy Reveals Disturbed Cerebral Capillary Blood Flow and Increased Susceptibility to Ischemic Insults in Diabetic Mice
Authors: Huang Ji-Yun , Liu Shuang-Shuang , Wilcox Christopher S , Lai En Y , Han Feng
Published: March 2022

Background:
Diabetes imposes an increased risk of microvascular complications. The present study aims to evaluate if diabetes and its disturbances in brain blood microcirculation increases the susceptibility to brain ischemic insults.

Methods and Results:
Diabetes was induced by streptozocin in mice expressing green fluorescent protein in endothelial cells (Tie2-GFP mice). Four weeks later the diabetic mice were subjected to transient (20 min) middle cerebral artery occlusion (MCAO).
In vivo
two-photon laser scanning microscopy (TPLSM) was applied to the brains of the mice, and blood flow was estimated in arteries, capillaries and veins in diabetic and control mice after the MCAO. We found a significant decrease in red blood cell (RBC) velocity in capillaries in diabetic mice (0.93 ± 0.11 mm/s,
P
<0.001 versus control mice). Despite this no significant changes in regional blood flow was observed by simultaneously performed laser-Doppler flowmetry. Notably, we observed that a further decline in RBC velocity (0.62 ± 0.07 mm/s,
P
<0.001 versus diabetic or ischemic mice) and capillary volume flux [(2.29 ± 0.27) × 10
-5
μl/s,
P
<0.001 versus diabetic or ischemic mice] in diabetic-MCAO mice, indicating diabetes-aggravated brain ischemia-induced disturbance in cerebral capillary blood flow. Furthermore, our intravital TPLSM data demonstrated that brain capillary flow was more turbulent in diabetic-MCAO mice than in the other groups. Diabetic-MCAO mice also exhibited increased neurological deficit (3.36 ± 0.23,
P
<0.01 versus MCAO mice), which was accompanied by exaggerated degradation of tight junction proteins and blunted CaMKII phosphorylation.

Conclusions:
The present data indicate that diabetic mice are more susceptible to disturbances of cerebral capillary blood flow and neurovascular defects during ischemic conditions.
Abstract 98: Myeloid-Specific Deletion of Prolyl Hydroxylase Domain Protein 2 Attenuates Hypertensive Cardiovascular Remodeling
Authors: Ichiki Toshihiro , Ikeda Jiro , Sunagawa Kenji
Published: March 2022

Background:
Hypertension induces cardiovascular hypertrophy and fibrosis. Infiltration of inflammatory cells including macrophages is critically involved in this process. We recently reported that inhibition of prolyl hydroxylase domain protein 2 (PHD2), which hydroxylates proline residue of hypoxia-inducible factor α (HIFα) and thereby induces HIFα degradation, suppressed inflammatory responses in macrophage. We examined whether myeloid-specific PHD2 deletion affects hypertension-induced cardiovascular remodeling.

Methods and Results:
Myeloid-specific PHD2-deficient mice (MyPHD2KO) were generated by crossing PHD2-floxed mice with LysM-Cre transgenic mice. HIF-1α and 2α proteins were accumulated and mRNA expression of TNFα, IL-6 and IL-1β, M1 macrophage markers, and TGF-β and connective tissue growth factor (CTGF) expression were significantly decreased in peritoneal macrophages from MyPHD2KO mice compared with those from control mice. PHD2-deficient macrophage showed attenuated migration toward MCP-1. Eight to 10 week-old mice were given L-NAME (30mg/kg), an eNOS inhibitor, dissolved in 0.9% NaCl in the drinking water for 14days. Angiotensin II (AngII, 0.8 mg/kg/day) was infused subcutaneously via an osmotic mini-pump for the last 7 days of the experiment. L-NAME/AngII comparably increased systolic blood pressure in control and MyPHD2KO mice. Cardiac interstitial fibrosis (3.4±0.4 vs. 2.3±0.4%, p<0.05), macrophage infiltration and cardiac hypertrophy induced by L-NAEM/AngII were ameliorated in MyPHD2KO mice. Echocardiography revealed that treatment with L-NAME/AngII induced left ventricular hypertrophy and reduction of ejection fraction in control mice, which were not observed in MyPHD2KO mice. L-NAME/AngII-induced expression of TGF-β, CTGF, and Collagen I was decreased in the heart of MyPHD2KO mice.

Conclusions:
Myeloid specific PHD2 deletion attenuates cardiac hypertrophy and fibrosis induced by L-NAME/AngII, which may be mediated by decreased inflammation- and fibrosis-associated gene expression in macrophage. PHD2 in myeloid lineage plays a critical role in hypertensive cardiovascular remodeling.
Abstract 002: Activation of AT1 receptors on Dendritic Cells Restrains Angiotensin II-Dependent Hypertension Through a CCR7-dependent Mechanism
Authors: zhang Jiandong , Karlovich Norah S , Griffiths Robert , crowley Steven D
Published: March 2022

Dendritic cells (DCs) trigger an adaptive immune response by presenting processed antigens to T lymphocytes. Given the emerging importance of adaptive immunity in hypertension, we posited that DCs may sample neoantigens in the kidney in order to prime pro-hypertensive T cells. Using CD11c-Cre mT/mG mice in which DCs fluoresce green, we detected robust green signals within the kidney after 2 wks of angiotensin (Ang) II-induced hypertension, confirming infiltration of DCs into the hypertensive kidney. As activation of type 1 angiotensin (AT1) receptors on other immune cell lineages has been shown to alter inflammatory responses, we explored the role of the DC AT1 receptor in blood pressure regulation by generating mice with DC-specific deletion of the AT1A receptor (CD11c Cre+ Agtr1aflox/flox = DC KO) and littermate controls (Agtr1aflox/flox = WT). Compared to WTs, the DC KOs had an ≈87% reduction in AT1A receptor mRNA expression in DCs (p<0.0001) but preserved AT1A expression in B and T lymphocytes, kidney, and heart (p=NS). Next, we infused uni-nephrectomized DC KO mice and wild-type (WT) controls with Ang II (500ng/kg/min) for 4 wks. At baseline, the mean arterial blood pressure (MAP) was similar in WT and DC KO mice (123±2 vs. 126±2 mm Hg; p=NS). By contrast, after the 1st week of Ang II infusion, DC KO mice had markedly exaggerated MAP elevations compared to WTs (174±4 vs. 158±5 mm Hg; p=0.026). Expression of the chemokine receptor CCR7 on mature DCs and T cells directs both cell lineages to the lymph node where the DC can display a processed antigen and activate the T cell. We detected higher levels of CCR7 mRNA in the kidneys from the Ang II-infused DC KOs compared to WTs (2.98±0.90 vs. 1.00±0.16 au; p=0.04), and posited that enhanced DC priming of T cells in the DC KOs contributed to their exaggerated hypertension. Consistent with this possibility, uni-nephrectomized CCR7-deficient (KO) mice had a significantly blunted chronic hypertensive response to Ang II (165±4 vs. 178±2 mm Hg; p=0.03) and ameliorated albuminuria (2.1±0.2 vs. 3.7±0.8 mg/24hrs; p=0.02) versus WT controls. We conclude from these data that stimulation of AT1 receptors on DCs constrains Ang II-induced blood pressure elevation, possibly by limiting the CCR7-dependent activation of T lymphocytes by DCs.
Abstract 005: Involvement of Decreases in Regulatory T Cells and Splenic Sympathetic Nerve in the Development of Hypertension in Genetically Hypertensive Rats
Authors: Katsuki Masato , Hirooka Yoshitaka , Kishi Takuya , Sunagawa Kenji
Published: March 2022

Background:
Autonomic nervous system can modulate immune system thereby influences the pathogenesis of hypertension. Among the immune cells, activated T cells have been implicated in the genesis of hypertension. In contrast, regulatory T cells (Treg cells), which maintain immune homeostasis, are reported to prevent AngII-induced hypertension. However, there are few studies of Treg cells in spontaneously hypertensive models. In particular, the spleen is an important organ to regulate immune system and splenic nerve can modulate immune responses in the spleen. Here, we investigated the role of Treg cells in hypertension and the involvement of splenic sympathetic nerve in genetically spontaneously hypertensive rats.

Methods and Results:
We evaluated Treg cells in stroke-prone spontaneously hypertensive rats (SHRSP) at 3 week (early pre-hypertensive stage), 5 week (late pre-hypertensive stage), or 15 week (established hypertensive stage) old, and in age-matched normotensive Wistar-Kyoto rats (WKY). CD4+CD25+Foxp3+ cells (Treg cells) in the spleen and peripheral blood did not differ between WKY and early pre-hypertensive SHRSP, whereas significantly decreased both in late pre-hypertensive (%CD25+Foxp3+ gated on CD4+ cells in the spleen; 7.7±0.5 vs. 5.5±0.3, n=5 for each, p=0.0080, blood; 6.7±0.7 vs. 3.4±0.8, n=4 for each, p=0.0060) and established hypertensive SHRSP (spleen; 9.9±0.7 vs. 5.4±0.4, n=4 for each, p=0.0011, blood; 8.8±0.7 vs. 4.5±0.1, n=4 for each, p=0.0009). We performed splenic denervation (or sham operation) in SHRSP at prehypertensive stage. After 4 weeks of splenic denervation, splenic Treg cells were increased compared with sham-operated SHRSP (4.2±0.1 vs. 6.1±0.3, n=7 for each, p<0.0001). Furthermore, splenic denervation in SHRSP at prehypertensive stage delayed blood pressure elevation (n=7 for each, p<0.001, vs. sham-operated SHRSP), whereas splenectomy did not affect blood pressure.

Conclusion:
Our results indicate that splenic denervation attenuates the decrease in splenic Treg cells and delays the development of hypertension, suggesting that splenic sympathetic nerve and decrease in Treg cells play a key role in the onset of hypertension in SHRSP.
Abstract 023: Neuronal Overexpression of the Human (Pro)renin Receptor Increases Sympathetic Tone that is Masked by Upregulation of Endothelial Nitric Oxide Synthase
Authors: Sullivan Michelle , Li Wencheng , Sigmund Curt D , Earley Scott , Feng Yumei
Published: March 2022

The binding of prorenin to the (pro)renin receptor (PRR) induces non-proteolytic activation of prorenin and generation of angiotensin II (Ang II). PRR activation can also induce Ang II-independent signaling pathways. However, whether the Ang II-independent signaling pathways are important for blood pressure (BP) regulation is not known. To address this question, we created transgenic mice that overexpress the human PRR (hPRR) selectively in neurons (syn-PRR mice). Activated human prorenin (hPRO) cannot cleave endogenous mouse angiotensinogen to generate Ang II. Therefore, administration of hPRO to syn-PRR mice can be used to examine Ang II-independent PRR signaling. Intracerebroventricular infusion of hPRO increases BP in syn-PRR (ΔMAP: 23 ± 4.6) but has no effect on WT mice (ΔMAP: 2 ± 0.8). The hPRO-induced pressor response in syn-PRR mice is unaffected by treatment with the Ang II type 1 receptor blocker losartan (ΔMAP: 19 ± 5.2), suggesting that hPRR activation increases BP independent of Ang II. Interestingly, although basal MAP (101 ± 1.5 vs. 101 ± 1.3) and HR (552 ± 3.3 vs. 544 ± 7.3) are similar between syn-PRR and wildtype (WT) mice (n = 8/group), syn-PRR mice have increased basal cardiac (ΔHR: -68 ± 1.3 vs. -45 ± 1.4) and vasomotor (ΔMAP: -41 ± 2.1 vs. -29 ± 0.4) sympathetic tone. We hypothesized that the vasodilator-generating enzyme endothelial nitric oxide synthase (eNOS) activity was augmented in the periphery to compensate for increased sympathetic activity. qPCR revealed that eNOS mRNA is increased in mesenteric arteries from syn-PRR vs. WT mice (expression ratio 1.54 vs. 1.0). To examine eNOS activity we inhibited NO production by systemic administration of the L-arginine analogue L-NAME. L-NAME (400 mg/kg) was injected daily for 7 days. L-NAME treatment resulted in an increase in BP (MAP: 125 ± 5.2) in WT mice that normalized by 24 hrs post-injection (MAP: 106 ± 6.7). Interestingly, L-NAME treatment in syn-PRR mice resulted in an elevation of BP (MAP: 131 ± 5.1) that was sustained 24 hrs post-injection (MAP: 123 ± 2.6), suggesting that NOS activity is increased in syn-PRR mice. Together, these data provide evidence that neuronal overexpression of hPRR increases sympathetic activity that is masked by the subsequent upregulation of eNOS in the periphery.
Abstract 024: 20-HETE Activates the Transcription of Endothelial Angiotensin Converting Enzyme (ACE) via NF-kB Translocation and Promoter Binding
Authors: Garcia Victor , Milhau Laura , Falck John R , Schwartzman Michal L
Published: March 2022

Increased vascular 20-hydroxyeicosatetraenoic acid (20-HETE), a cytochrome P450-arachidonic acid metabolite, promotes vascular dysfunction, injury and hypertension that is dependent, in part, on the renin angiotensin system (RAS). We showed that in cultured human microvascular endothelial cells (HMVEC), 20-HETE (5 nM) increases angiotensin converting enzyme (ACE) mRNA, protein, and cellular and extracellular ACE activity via EGFR-tyrosine kinase-MAPK-IKKβ- signaling pathway. Here we show that treatment of HMVEC with EGF (100 ng/ml) alone or with 20-HETE (10 nM) did not induce or further increase ACE mRNA levels. Pretreatment of cells with a neutralizing antibody against EGF did not prevent the 20-HETE- or 20-HETE+EGF-mediated ACE induction, indicating that the induction of ACE by 20-HETE is EGF-independent. The NF-kB inhibitor JSH-23 prevented the 4.58-fold (±0.78; p<0.05) 20-HETE (10nM)-mediated induction of ACE. EMSA of 20-HETE-treated cells showed increased NF-kB binding activity in nuclear extracts. In cells transfected with luciferase promoter-reporter constructs for the somatic or germinal ACE promoter regions, 20-HETE (10 nM) increased promoter activity by 4.37-fold (±0.18; p<0.05) and 2.53-fold (± 0.24; p<0.05), respectively. 20-HETE-stimulated ACE promoter activity was abrogated by the 20-HETE antagonist 20-HEDE and by inhibitors of EGFR, MAPK, IKKβ and NF-kB activation. Sequence analysis demonstrated the presence of two (Chr 17: S1: 61554061-61554075 , S2: 61554157-61554171) and one (Chr 17: S3: 61562250-61562264) putative NF-kB binding sites on the human somatic and germinal ACE promoters, respectively. CHIP assay indicated that 20-HETE stimulates the translocation and subsequent binding of NF-kB to each of the putative binding sites (S1: 3.43±0.3, S2: 3.72±0.68, S3: 3.20±0.18-fold enrichment vs vehicle; p<0.05). In vivo, increased vascular 20-HETE levels are associated with increased phospho-IKK and ACE levels that are prevented by treatment with 20-HETE antagonists. This is the first study to identify NF-kB as a transcriptional factor for ACE and to implicate a distinct EGFR/MAPK/IKK/NF-kB signaling cascade underlying 20-HETE-mediated transcriptional activation of ACE mRNA and stimulation of ACE activity.
Abstract 030: Dpp4 Inhibition Substantially Reverses Ang II-mediated Inflammation And Kidney Injury Independent Of BP Reduction.
Authors: Nistala Ravi , Aroor Annayya , Meuth Alex , Sowers James , Whaley-Connell Adam , Bender Shawn , Mckarns Susan
Published: March 2022

Objectives:
DPP4 is thought to play an important role in co-stimulation of T-lymphocytes and activation of DPP4 is thought to play a role in hypertension both via degradation of glucagon like peptide-1 (GLP-1) and via direct mechanisms. However, the triggers for DPP4 activation in hypertension are largely unknown except for one study showing insulin-mediated release of DPP4 from adipocytes. Recently, our lab demonstrated increased DPP4 activity in the plasma and kidney tissue of a Western diet-fed C57Bl/6 mouse that had elevated BP, proteinuria and inflammation with M2 to M1 macrophage polarization. In the same model, we observed activation of kidney renin-angiotensin system (unpublished data). Additionally, we observed that Ang II (angiotensin II) is able to increase DPP4 activity in proximal tubule cells
in vitro
(PTCs, (unpublished data). Therefore, we hypothesized that Ang II activation of DPP4 in mice will lead to inflammation in the kidney and albuminuria.

Methods:
Pressor dose Ang II (1000ng/kg/min) was infused into C57Bl/6 mice for 3 wks and BP was measured in conscious mice by radiotelemetry. Kidney tissue was analyzed via DPP4 activity assay, FACS, Western Blot, light and electron microscopy.

Results:
Ang II infused mice were characterized by elevated BP and albuminuria (4.5 fold) concomitant with mesangial widening and PTC injury. We also observed an increase in CD4+CD44
high
CD62L
low
(1.5 fold), decrease in CD4+FoxP3 (1.5 fold) concomitant with increased expression of DPP4 on CD3+CD4+ T-cells. Saxagliptin (10mg/kg/day) administered in peanut butter starting at 1 wk after Ang II, decreased DPP4 activity (~50%) in the plasma and kidney tissue. Saxagliptin had no effect on BP and albuminuria although there was amelioration of mesangial widening and PTC injury. Saxagliptin also mitigated DPP4 expression on CD3+CD4+ T-cells and reversed the changes in memory T-cells and Tregs.

Conclusions:
Ang II mediates BP increase and albuminuria via modulation of adaptive immunity which in turn may be regulated by DPP4. Saxagliptin reverses DPP4-mediated modulation of adaptive immunity and ameliorates kidney injury partially although it is unable to reverse established increases in BP and albuminuria.
Abstract 031: Novel Role of Nlrp3 Inflammasome Activation beyond Canonical Inflammation: Coronary Endothelial Injury and Repair
Authors: Zhang Yang , Xia Min , Chen Yang , Pitzer Ashley L , Li Xiang , Boini Krishna M , Li Pin-Lan
Published: March 2022

Inflammasomes are intracellular machinery turning on inflammatory response to danger factors. Recent studies reported that activation of inflammasomes also directly produce injurious action on cells or tissues independent of canonical inflammation. The present study hypothesized that activation of endothelial Nlrp3 inflammsome directly induces endothelial dysfunction at early stage of obesity without canonical inflammation yet. It was demonstrated that Nlrp3+/+ mice under 6-week high fat diet (HFD) had increased formation and activation of Nlrp3 inflammasome in coronary arterial endothelium compared to normal diet (ND)-treated mice, as shown by confocal microscopic detection of inflammasome aggregation and active caspase-1 formation. Ultrasound Doppler measurement showed that HFD markedly reduced bradykinin-induced increases in coronary blood flow by 95% in Nlrp3+/+ mice, but only by 45% in Nlrp3-/- mice. Vascular reactivity studies confirmed that HFD impaired endothelium-dependent vasodilation to acetylcholine (10-9-10-5 M) in freshly isolated coronary arteries of Nlrp3+/+ mice (maximum dilation: 53±3% vs. 84±3% of ND), but not in Nlrp3-/- mice (maximum dilation: 81±3% vs. 85±2% of ND). Similarly, visfatin (an injurious adipokine) simulates HFD-induced injurious action on endothelial dysfunction in normal Nlrp3+/+coronary arteries, which was attenuated by Nlrp3 gene deletion. In Nlrp3+/+ mice, HFD markedly increased the number of circulating endothelial progenitor cells (EPCs) in peripheral blood (1.73±0.69% vs. 0.46±0.05% of ND) and adhesion molecule VCAM-1 levels in coronary arterial endothelium. Nlrp3-/- mice fed HFD exibited higher level of circulating EPCs (4.57±1.27% vs. 0.32±0.04% of ND), but similar VCAM-1 expression. Together, these results suggest that endothelial Nlrp3 inflammasome activation in response to obesity or injurious adipokine produces endothelial dysfunction and inhibits EPC mobilization for endothelial repair, which are distinct from its canonical inflammatory responses such as inflammtory cell recruitment and infiltration in the vasculature. It is proposed that Nlrp3 inflammasome activation may trigger endothelial injury resulting in ultimate vascular inflammation and schlerosis.
Abstract 039: Oral Delivery of Angiotensin Converting Enzyme2 and Angiotensin-(1-7) Bioencapsulated in Plant Cells Attenuates Pulmonary Hypertension
Authors: Shenoy Vinayak , Chul Kwon Kwang , Rathinasabapathy Anandharajan , Lin Shina , Jin Guiying , Song Chunjuan , Shil Pollob , Nair Anand , Qi Yanfei , Li Qiuhong , Francis Joseph , Katovich Michael , Daniell Henry , Raizada Mohan
Published: March 2022

Rationale:
Emerging evidences indicate that diminished activity of the vasoprotective axis of the renin-angiotensin system, constituting angiotensin converting enzyme2 (ACE2) and its enzymatic product, angiotensin-(1-7) [Ang-(1-7)] contribute to the pathogenesis of pulmonary hypertension (PH). However, long-term repetitive delivery of ACE2 or Ang-(1-7) would require enhanced protein stability and ease of administration to improve patient compliance.

Objective:
To develop a low-cost oral delivery system for ACE2 and Ang-(1-7), and demonstrate their therapeutic efficacy in an experimental model of PH.

Methods:
We have taken advantage of transplastomic technology, which enables chloroplasts to generate high levels of therapeutic proteins within plant leaves. Transplastomic plants expressing ACE2 or Ang-(1-7) were created using biolistic delivery of chloroplast vectors. Male rats were given a single subcutaneous injection of monocrotaline (MCT, 50mg/Kg) to induce PH. Oral gavage of the frozen powdered leaves (500mg in phosphate buffered saline) expressing either ACE2 or Ang-(1-7) was performed twice daily for 4-weeks in MCT rats.

Results:
MCT injected rats exhibited marked increase in right ventricular systolic pressure (RVSP; Control: 33±1mmHg; MCT: 89±4mmHg),signifying development of PH, which was associated with right ventricular hypertrophy (RVH; Control: 0.28±0.01; MCT: 0.63±0.03).However, oral feeding of MCT rats with bioencapsulated ACE2 or Ang-(1-7) showed significant decreases in RVSP (MCT+ACE2: 55±8mmHg, MCT+Ang-(1-7): 62±7mmHg ) and RVH (MCT+ACE2: 0.39±0.05, MCT+Ang-(1-7): 0.4±0.03). Similarly, echocardiography data revealed significant improvement in right heart function, along with enhanced pulmonary blood flow in MCT animals fed with ACE2 or Ang-(1-7). Furthermore, histological studies revealed considerable reduction in right ventricular fibrosis and pulmonary vessel wall thickness in ACE2 or Ang-(1-7) fed animals.These protective effects were associated with decreased inflammation.

Conclusion:
Our study provides the first proof-of-concept for a novel low-cost oral ACE2 and Ang-(1-7) delivery system, using transplastomic technology that has significant clinical implications for treating PH.
Abstract 070: Superoxide And Hydrogen Peroxide Differentially Modulate Mouse Afferent Arteriolar Myogenic Responses Via Protein Kinase C And Protein Kinase G And Their Coupling To Membrane Potential
Authors: Li Lingli , Lai Eny Yin , Wilcox Christopher S
Published: March 2022

Background:
Although reactive oxygen species (ROS) mediate normal myogenic responses (MRs) of afferent arterioles (Affs), oxidative stress contribute to impaired MRs in chronic kidney disease (CKD) and thereby to barotrauma. The mechanisms underlying these apparently conflicting reports are unclear. MRs depend on reductions in membrane potential (Em) that activate voltage-operated calcium channels to raise intracellular [Ca
2+
]. Therefore, we tested the hypothesis that MRs are increased by superoxide (O
2
.-
) and reduced by hydrogen peroxide (H
2
O
2
) through differential signaling via protein kinase C (PKC) - and protein kinase G (PKG) - dependent changes in Em.

Methods:
MRs were assessed from changes in luminal diameter of mouse individual isolated perfused Affs (n = 5-6 per group) with change in perfusion pressure (PP) from 40 to 80 mmHg. The effects of O
2
.-
and H
2
O
2
on the MR were assessed after incubation with 1 μM paraquat (PQ increases O
2
.-
) or incubation with 10 μM H
2
O
2
. O
2
.-
with PP was assessed from PEG-SOD inhibitable ethidium:dihydroethidium fluorescence, and H
2
O
2
from PEG-catalase (CAT) inhibitable H
2
DCFDA fluorescence and Em from DiBAC
4
(3) fluorescence.

Results:
1. PQ increased O
2
.-
with PP by 32 ± 4% (P<0.001) and MR by 140 ± 33% (P<0.01) (prevented by PEG-SOD), but H
2
O
2
reduced MR by 50 ± 2% (P<0.0001) (prevented by PEG-CAT). 2. The PP-induced fall in Em was increased by PQ (PQ -19 ± 2% vs vehicle -4 ± 1%, P<0.001), but was reversed by H
2
O
2
(H
2
O
2
+16 ± 1% vs vehicle -4 ± 1%, P<0.001). 3. Inhibition of PKC with 3 μM of Gö6983 blocked the reduction in Em with PQ (PQ -19 ± 2% vs PQ+ Gö6983 -2 ± 2%, P<0.01), while inhibition of PKG with 30 μM of Rp-8-Br-PET-cGMPS blocked the increase in Em with H
2
O
2
(H
2
O
2
+16 ± 2% vs H
2
O
2
+Rp-8-Br-PET-cGMPS -2 ± 1%, P<0.05).

Conclusions:
O
2
.-
signals via PKC to depolarize the Em and thereby to activate the MR whereas H
2
O
2
signals via PKG to hyperpolarize the Em and thereby to inhibit the MR. Thus, O
2
.-
and H
2
O
2
signaling via specific G-protein-coupled changes in Em may explain the differential effects of ROS on MRs.
Abstract 079: (pro)renin Receptor Knockdown In The Paraventricular Nucleus Of Hypothalamus Attenuates The Development Of Doca-salt Hypertension
Authors: Li Wencheng , Feng Yumei
Published: March 2022

We previously reported that neuron-specific PRR deletion prevents the development of deoxycorticosterone acetate (DOCA)-salt induced hypertension. However, it is not clear which brain region is responsible for the effect of PRR knockout on the development of hypertension. In present study, we bilaterally micro-injected adeno-associated virus (AAV2)-mediated Cre recombinase with an eGFP tag (AAV-Cre, 50 nl/side) into the paraventricular nucleus of hypothalamus (PVN) of PRR-Floxed mice to knockdown the PRR expression in this region. Deoxycorticosterone acetate-salt (DOCA, 50mg, 0.9%NaCl drinking solution, 21 days) treatment was started 3 days after AAV-Cre or control virus (AAV-eGFP) injection. Blood pressure (BP) was monitored using radio telemetric probes in conscious animals. Autonomic functions were also assessed using pharmacological methods. PVN was micro-punched for PRR real time PCR to verify PRR knockdown efficiency. Three days after virus injection, AAV-Cre significantly decreased PRR expression (fold change: 0.69 ± 0.1 vs. 1.0 ± 0.05) in the PVN compared to AAV-eGFP before DOCA-salt treatment. Three weeks after DOCA-salt treatment, PRR mRNA levels were increased in the AAV-eGFP group (fold change: 3.0 ± 0.1), and this increase was abolished by AAV-Cre (fold change: 1.2 ± 0.04, P<0.05). AAV-Cre expression in the PVN had no effect on PRR expression in the cerebral cortex or brain stem. Blood pressure (mmHg) and heart rate (HR, bpm) were recorded at baseline and following DOCA-salt treatment. AAV-Cre or control virus had no effect on baseline BP or HR. Following DOCA-salt treatment, BP was significantly increased in mice treated with AAV-eGFP (138.5 ± 7.4); while BP was significantly lower in AAV-Cre treated mice (118.9 ± 3.4). The cardiac sympathetic tone (ΔHR to propranolol: -123 ± 46 vs. -181 ± 16) and vasomotor sympathetic tone (ΔBP to chlorisondamine: -47 ± 9.2 vs. -69 ± 4.7) were significantly lower in AAV-Cre treated mice after DOCA-salt treatment. The cardiac parasympathetic tone (ΔHR to methylatropine: 82 ± 18 vs. 40 ± 7.3) was greater in AAV-Cre treated mice. In summary, PRR knockout in the PVN attenuates the development of DOCA-salt hypertension and is associated with the improvement of autonomic function.
Abstract 091: Chronic Vasopressin Infusion: A Novel, Clinically Significant, and
Pregnancy-Specific
Mouse Model of Preeclampsia
Authors: Santillan Mark , Santillan Donna A , Scroggins Sabrina M , Min James Y , Sangren Jeremy A , Pearson Nicole A , Gibson-Corley Katherine , Grobe Justin L
Published: March 2022

Recently we demonstrated that the late-pregnancy cardiovascular disorder, preeclampsia, is characterized by robust, early and sustained vasopressin (AVP) hypersecretion in all three trimesters of pregnancy in humans. We hypothesize a causative role for elevated AVP in the pathogenesis of preeclampsia. This concept was tested by chronically infusing AVP (0.24 - 240 ng/hr, s.c., or saline vehicle) in wildtype C57BL/6J female mice throughout pregnancy. In pregnant mice, AVP infusion caused
pregnancy-specific
increases in systolic blood pressure by tail-cuff plethysmography with significant increases in the pregnant cohort (saline n=16: 110±3, vs 24 ng/hr AVP n=11: 120±3 mmHg on GD15/16, P<0.05), but no significant difference in the nonpregnant cohort (saline n=14: 108±3, vs 24 ng/hr AVP n=5: 108±5 mmHg, P>0.05). In addition, the AVP infused mice exhibited increased proteinuria (0.24 ng/hr n=2: 66±73, 2.4 ng/hr n=5: 348±56, 24 ng/hr n=2: 799±297 mg/d, P<0.05), intrauterine growth restriction (saline n=25: 0.78±0.06, vs 24 ng/hr n=65: 0.55±0.03 g/fetus, P<0.05), spontaneous feto-placental unit resorption (saline: 0/65=0%, 0.24 ng/hr: 0/16=0%, 2.4 ng/hr: 2/45=4%, 24 ng/hr 8/73=11%, P<0.05), and maternal renal glomerular endotheliosis by electron microscopy. High doses of AVP reduced rates of successful pregnancy with single-night breeding (saline 8/18=44% vs 240 ng/hr 1/20=5%, P<0.05). These data demonstrate that AVP infusion - which simulates the large sustained increases in AVP secretion during human preeclampsia - is sufficient to induce all the cardinal phenotypes of preeclampsia in pregnant C57BL/6J mice. This identifies AVP infusion as a novel, clinically significant, and
pregnancy-specific
physiological model of preeclampsia in mice. These data support our hypothesis that AVP hypersecretion during pregnancy may be causative for the development of preeclampsia. Ongoing experiments are aimed at identifying the causes of AVP hypersecretion in human preeclampsia, the target tissues and receptors involved, and the utility of targeting AVP signaling as a novel therapeutic for preeclampsia.
Abstract 100: Interference with Smooth Muscle Peroxisome Proliferator-Activated Receptor-gamma (PPARG) Exacerbates Hypertension and Vascular Dysfunction: Role of TIMP-4
Authors: Ketsawatsomkron Pimonrat , Davis Deborah R , Grobe Justin L , Keen Henry L , Sigmund Curt D
Published: March 2022

PPARG, a ligand-activated transcription factor plays a critical role in the regulation of blood pressure and vascular function. We hypothesized that interference of smooth muscle cell (SMC) PPARG exacerbates hypertension (HT) and resistance vessel dysfunction. Transgenic mice expressing dominant negative PPARG (S-P467L) in SMC or non-transgenic controls (NT) were implanted with DOCA pellet and allowed ad libitum access to 0.15 M NaCl for 21 days in addition to regular chow and water. Blood pressure was monitored by telemetry and mesenteric arterial (MA) function was assessed by pressurized myograph. At baseline, 24-hour mean arterial pressure (MAP) was similar between NT and S-P467L mice. DOCA-salt increased MAP to a much greater degree in S-P467L mice (Δ MAP; S-P467L: +34.2±6.0, NT: +13.3±5.7, p<0.05 vs NT). Vasorelaxation to acetylcholine was slightly impaired in S-P467L MA compared to NT at baseline whereas this effect was further exaggerated after DOCA-salt (% relaxation at 10-5 M, S-P467L: 56.1±8.3, NT: 79.4±5.6, p<0.05 vs NT). Vascular morphology at luminal pressure of 75 mmHg showed a significant increase in wall thickness (S-P467L: 18.7±0.8, NT: 16.0±0.4, p<0.05 vs NT) and % media/lumen (S-P467L: 8.4±0.3, NT: 7.1±0.2, p<0.05 vs NT) in S-P467L MA after DOCA-salt. Gene expression profiling in MA revealed a loss of the tissue inhibitor of metalloproteinases (TIMP)-4 in S-P467L compared to NT at baseline (fold change compared to NT; 0.39±0.2, p<0.05). Interestingly, expression of TIMP-4 was significantly increased in MA of NT treated with DOCA-salt compared to NT baseline (1.5±0.1 fold). However, the induction of TIMP-4 was lost in S-P467L with DOCA-salt. There was no difference in mRNA expression of other TIMPs between NT and S-P467L after DOCA-salt. Inhibition of PPARG in primary SMC cultures either by pharmacological inhibitor, GW9662 or mutant PPARG resulted in down-regulation of TIMP-4 expression. Moreover, GW9662 treatment led to an increased total matrix metalloproteinases activity in SMC. We conclude that loss of PPARG function sensitizes the mice to the effects of DOCA-salt induced HT and vascular dysfunction, in part, by preventing the normal protective up-regulation of TIMP-4.