Mouse Model Of Hypertension Research Articles

Interference of MDM2 attenuates vascular endothelial dysfunction in hypertension partly through blocking Notch1/NLRP3 inflammasome pathway

BackgroundHypertension is a life-threatening disease mainly featured as vascular endothelial dysfunction. This study aims to explore the regulatory role of murine double minute 2 (MDM2) in hypertension and vascular damage. MethodsMice were infused with angiotensin II (AngII) to establish a hypertension mouse model in vivo and AngII-stimulated HUVECs were constructed to simulate the damage of vascular endothelial cells in hypertension in vitro. The plasmids targeting to MDM2 was injected to mice or transfected to HUVECs. qRT-PCR and western blot were performed to detect corresponding gene expression in mice aorta. Blood pressure was measured. H&E and Masson staining were conducted to evaluate histological changes of aorta. Responses to the acetylcholine (ACh) and sodium nitroprusside (SNP) were assessed in aorta. ZO-1 expression and cell apoptosis were detected by immunofluorescence and TUNEL, respectively. Network formation ability was determined employing a tube formation. ResultsMDM2 was upregulated in hypertensive mice. Knockdown of MDM2 inhibited AngII-induced high BP, histological damage, vascular relaxation to Ach, and promoted the levels of p-eNOS and ZO-1 in the aorta in hypertensive mice. MDM2 knockdown inactivated Notch1 signaling and NLRP3 inflammasome, while the inhibitory effect of MDM2 knockdown on NLRP3 inflammasome activation was partly restored by the activation of Notch1. Furthermore, knockdown of MDM2 relieved AngII-induced endothelial dysfunction in HUVECs, as well as suppressing AngII-promoted cell apoptosis. Whereas, the impacts generated by MDM2 knockdown were partly weakened by the activation of Notch1 signaling or NLRP3 inflammasome. ConclusionIn summary, knockdown of MDM2 can attenuate vascular endothelial dysfunction in hypertension, which may be achieved through inhibiting the activation of Notch1 and NLRP3 inflammasome.

Beneficial Role of Erythrocyte Sphingosine 1-Phosphate in Preventing Renal Macrophage Polarization and Fibrosis through Combating HIF-1α-Dependent Creatine Phosphate Shuttle

Introduction: Erythrocytes, the major reservoir and supplier for plasma sphingosine 1 phosphate (S1P), is a potent biolipid signaling via its specific receptors responsible for immune cell trafficking and function. Current evidence has revealed that erythrocyte intracellular S1P has its own function to promote oxygen (O 2) delivery to counteract tissue hypoxia. Aim: To understand whether O 2 delivery mediated by erythrocyte S1P is involved in tissue macrophage polarization and fibrosis. Methods and Results: Genetic ablation of erythrocyte SphK1 (the only enzyme to generate S1P in erythrocyte, eSphK1 -/-) resulted in less O 2 delivery from erythrocytes, thus leading to severe renal hypoxia, damage and fibrosis in angiotensin II induced hypertensive kidney disease mouse model. Surprisingly, although plasma S1P level was reduced to 50% in Ang II-infused eSphK1 -/- mice, these mice displayed preferentially elevated pro-fibrosis M2 macrophages surrounding the hypoxic renal tubules. Surprisingly, there was no significant difference of S1P level in either kidney or renal macrophages between Ang II-infused controls and eSphK1 -/- mice, ruling out the possibility of difference of kidney or macrophage S1P levels responsible for renal M2 macrophage polarization. Metabolically, untargeted high-throughput metabolic profiling in macrophages purified from mouse kidney revealed a substantial reduction of amino acids (AAs) and fatty acids (FAs) but increased creatine. We further validate this in vitro by 13C 6, 15N 4-labeled arginine tracer. Hypoxia (1~2% O 2) promoted increased 13C 2-labeled creatine (arginine downstream metabolite) in murine bone marrow derived macrophages (BMDMs) time dependently. At cellular and molecular level, we further demonstrated that hypoxia signaling via HIF-1a directly enhanced IL-4 induced M2 polarization of BMDMs as well as expression of Ckb (a gene encoding creatine shuttle), which can be reversed by Chrysin (an HIF-1α inhibitor). Preclinical studies revealed that chrysin rescued the severe renal damage in eSphK1 -/-mice manipulated with unilateral ureteral obstruction (UUO) and prevented development of chronic kidney disease (CKD) in the control mice. Translational research validated mouse findings in kidney tissue of CKD patients. Conclusion: Our work has revealed that erythrocyte S1P combats HIF1α-dependent creatine shuttle, renal macrophage polarization and fibrosis and highlighted potential diagnostic and therapeutic possibilities for macrophage polarization and renal fibrosis.

A programmable protease-based protein secretion platform for therapeutic applications.

Cell-based therapies represent potent enabling technologies in biomedical science. However, current genetic control systems for engineered-cell therapies are predominantly based on the transcription or translation of therapeutic outputs. Here we report a protease-based rapid protein secretion system (PASS) that regulates the secretion of pretranslated proteins retained in the endoplasmic reticulum (ER) owing to an ER-retrieval signal. Upon cleavage by inducible proteases, these proteins are secreted. Three PASS variants (chemPASS, antigenPASS and optoPASS) are developed. With chemPASS, we demonstrate the reversal of hyperglycemia in diabetic mice within minutes via drug-induced insulin secretion. AntigenPASS-equipped cells recognize the tumor antigen and secrete granzyme B and perforin, inducing targeted cell apoptosis. Finally, results from mouse models of diabetes, hypertension and inflammatory pain demonstrate light-induced, optoPASS-mediated therapeutic peptide secretion within minutes, conferring anticipated therapeutic benefits. PASS is a flexible platform for rapid delivery of therapeutic proteins that can facilitate the development and adoption of cell-based precision therapies.

FRI141 Dietary Salt Regulates mTORC1 Signaling In Humans And Mice With Hypertension

Abstract Disclosure: H. Aljaibeji: None. D.L. Brooks: None. A.E. Garza: None. P. Gerges: None. M. Heydarpour: None. P. Luminita: None. J.S. Williams: None. G. Williams: None. J.R. Romero: None. Raptor and the mammalian target of rapamycin (mTOR) form a complex (mTORC1) that has been proposed to play a role in aldosterone (ALDO) production. Pharmacological inhibition of mTORC1 reduces blood pressure (BP) and ALDO levels. However, the effects of environmental factors such as dietary salt on mTORC1 in humans are unclear. We studied the impact of one-week liberal Na+ (LIB, 200 mmol/day) and restricted (RES, 10 mmol/day) Na+ diets on Raptor and mTOR expression in human peripheral blood mononuclear cells (PBMC) from hypertensive subjects. Raptor and mTOR expression were higher on PBMCs from subjects on a LIB than on RES Na+ diet (n=13; P=0.036 and P=0.0008, respectively). We also studied Caucasian subjects (n=792) from the Hypertensive Pathotype (HyperPATH) cohort using the Multi-Ethnic Genotyping Array on the Illumina platform. Candidate gene association analyses showed that the single nucleotide polymorphic (SNP) Raptor gene variant, rs99001846, was associated with BP and ALDO levels. Multivariate linear regression analyses performed on hypertensive and normotensive participants showed that in hypertensives, rs99001846 risk allele carriers (AA/GA), as compared to non-risk carriers (GG), were associated with increased systolic BP levels on bothLIB and RES Na+ diets (P=0.02, P=0.01 respectively). In addition, risk allele carriers, as compared to non-risk carriers, showed increased ALDO and reduced Na+ excretion on a LIB Na+ diet (P=0.009, P=0.025, respectively). We also studied a mouse model of low nitric oxide and high AngII-mediated hypertension and observed increased cardiac Raptor and mTOR expression (n=4, P=0.0008 and P=0.03 respectively). We then performed in silico bioinformatic analyses of rat zona glomerulosa cells following 2 hr treatment with 100 nM angiotensin II (AngII) using the Gene Expression Omnibus (GEO). AngII treatment led to the activation of mTOR signaling pathways as shown by increased expression of insulin receptor substrate type 2 (P=0.00001) and Low-density lipoprotein-related receptor 5 (P=0.03). Also, AngII reduced the expression of Eukaryotic Translation Initiation Factor 4E Binding Protein 1 by 43% (P= 0.001) and increased expression of CAP-Gly Domain Containing Linker Protein 1 by 70% (P= 0.00003) - important signaling molecules that are downstream of mTOR activation. Our results suggest that in hypertension, Na+ intake and Raptor gene variants modify mTORC1. Thus, mTOR/Raptor expression are a potential biomarker of ALDO-mediated cardiovascular injury. Presentation: Friday, June 16, 2023

In silico analysis and verification of critical genes related to vascular calcification in multiple diseases.

Identifying a functional molecular therapeutic target of vascular calcification (VC) that will not affect normal osteogenic differentiation is a challenge. To address this aim, we screened the differentially expressed genes (DEGs) in different VC conditions, including endothelial-osteogenic transition (EOT) (GSE167962), chronic kidney disease (CKD), and atherosclerosis (AS) (GSE159832). KEGG pathways, protein-protein interactions, and hub genes were also analyzed. The intersecting DEGs among the EOT, CKD, and AS groups were verified by quantitative reverse transcription polymerase chain reaction and immunohistochemistry in a DOCA-salt hypertension mouse model. The phosphoinositide 3-kinase-protein kinase B signaling pathway, ECM-receptor interaction, chemokine signaling pathway, and focal adhesion were enriched in EOT and AS-induced VC. ECM-receptor interaction, PPAR signaling pathway, apelin signaling pathway, AMPK signaling pathway, adipocytokine signaling pathway, and cholesterol metabolism were enriched in CKD and AS-induced VC. C4b, Cebpa, Lyz2, and Spp1 were also upregulated in EOT, CKD, AS, and hypertension. This study identified promising molecular targets for VC therapy.

High-Soluble-Fiber Diet Attenuates Hypoxia-Induced Vascular Remodeling and the Development of Hypoxic Pulmonary Hypertension.

Hypoxic pulmonary hypertension is a difficult disease to manage that is characterized by sustained elevation of pulmonary vascular resistance and pulmonary artery pressure due to vasoconstriction, perivascular inflammation, and vascular remodeling. Consumption of soluble-fiber is associated with lower systemic blood pressure, but little is known about its ability to affect the pulmonary circulation. Mice were fed either a low- or high-soluble-fiber diet (0% or 16.9% inulin) and then exposed to hypoxia (FiO2, 0.10) for 21 days to induce pulmonary hypertension. The impact of diet on right ventricular systolic pressure and pulmonary vascular resistance was determined in vivo or in ex vivo isolated lungs, respectively, and correlated with alterations in the composition of the gut microbiome, plasma metabolome, pulmonary inflammatory cell phenotype, and lung proteome. High-soluble-fiber diet increased the abundance of short-chain fatty acid-producing bacteria, with parallel increases in plasma propionate levels, and reduced the abundance of disease-related bacterial genera such as Staphylococcus, Clostridioides, and Streptococcus in hypoxic mice with parallel decreases in plasma levels of p-cresol sulfate. High-soluble-fiber diet decreased hypoxia-induced elevations of right ventricular systolic pressure and pulmonary vascular resistance. These changes were associated with reduced proportions of interstitial macrophages, dendritic cells, and nonclassical monocytes. Whole-lung proteomics revealed proteins and molecular pathways that may explain the effect of soluble-fiber supplementation. This study demonstrates for the first time that a high-soluble-fiber diet attenuates hypoxia-induced pulmonary vascular remodeling and the development of pulmonary hypertension in a mouse model of hypoxic pulmonary hypertension and highlights diet-derived metabolites that may have an immuno-modulatory role in the lung.

Abstract P370: Progranulin Regulates Vascular Function And Blood Pressure Dependent On Ephrin A2 And Sortlin1 Receptors And Nitric Oxide Production.

Background: Progranulin (PGRN) is implicated in scar formation, cell proliferation, and anti-inflammation. Although high level of circulating PGRN was previously reported in hypertension, it is still not clear if PGRN can counteract the increases in blood pressure (BP) via modulating vascular tone. We sought to examine the role of PGRN maintaining the vascular tone and BP. Methods: we used male and female global PGRN deficient mice (PGRN-/-) and their counterparts (PGRN+/+) of 10-12-weeks of age to study vascular function and BP by wire myograph and radiotelemetry, respectively. Results: We found that two different mouse models of hypertension (angiotensin II or aldosterone treatments), elevated the levels of circulating PGRN. We also found that male and female PGRN-/- mice display elevated mean arterial pressure (MAP) (mmHg: PGRN+/+: 102.3 ± 3.9 vs PGRN-/-: 112.9 ± 3.7*, *P<0.05) followed by vascular hypercontractility to noradrenaline (NA) [Maximal response (% of 60mM of KCl) male PGRN+/+: 121.9 ± 14.1 PGRN-/-: 152.6 ± 18.8*; female PGRN+/+: 102.6 ± 4.1 and PGRN-/- 131.3 ± 11.4*, *P<0.05) in mesenteric arteries (MA), and increased circulating CCL2, IL-13, and interferon-γ. Whereas replacing PGRN with recombinant PGRN (rPGRN, 20ug/day/7-days) restored the MAP and vascular contractility in PGRN-/-. To explore the mechanisms by which PGRN modulates vascular tone, we treated MA from PGRN+/+, 1-hour prior NA curves, with rPGRN (600ng/mL) and found that PGRN attenuates the vascular contractility, such effect was abolished by blocking Ephrin A2 (EphA2) and Sortlin1 (Sort1) receptors or nitric oxide (NO) synthase (NOS). Next, we freshly isolated endothelial cells (EC) from mesentery of male and female PGRN+/+ mice via magnetic CD31+ microbeads to evaluate any sex difference on EphA2 and Sort1 expressions, which revealed that both receptors are equally expressed in EC from both sexes. In isolated mouse mesenteric EC, rPGRN stimulated NO formation and endothelial NOS activation, which were prevented by blocking EphA2 and Sort1. Conclusion: PGRN adjusts the vascular tone and BP via modulating NO formation and EphA2 and Sort1 receptors activation. Therefore, PGRN might be elevated in hypertension as a tentative to reduce BP by regulating vascular resistance.

Abstract P188: Hypertensive Stimuli Increase Pro-Inflammatory Bone Marrow Derived Macrophages In Vitro

Renal macrophage infiltration and inflammation is a hallmark of hypertension (HTN). An imbalance in macrophages has been observed in murine salt-sensitive and angiotensin II (AngII)-induced models of HTN. We have identified increased activated macrophages (CD45+ CD11b+ F4/80+ CD38+), pro-inflammatory M1 macrophages (CD45+ CD11b+ F4/80+ CD11c+ CD206-), and activated M1 macrophage (CD45+ CD11b+ F4/80+ CD11c+ CD206- CD38+) in the kidneys of these mice. In this study, we hypothesized that salt and AngII directly induce macrophages to become activated and pro-inflammatory. We exposed bone marrow derived macrophages (BMDMs) grown in GM-CSF to salt (190 μm) and AngII (0.01 μm) and found that there were significant increases in renal activated macrophages (% of CD45 cells: Con: 31 ± 1; AngII: 37 ± 1; Salt: 56 ± 1), pro-inflammatory M1 macrophages (Con: 27 ± 1; AngII: 37 ± 1; Salt: 56 ± 1), and activated M1 macrophages (Con: 22 ± 1; AngII: 28 ± 1; Salt: 43 ± 1) as determined by flow cytometry. However, the cultivation of BMDMs in M-CSF, the more traditional growth factor present in culturing BMDMs, did not produce the same significant results. Next, we will identify renal pro-inflammatory cytokines, chemokines, growth factors, and levels of GM-CSF present in these mouse models of HTN. Currently, these findings suggest that GM-CSF increases macrophage responsiveness to HTN stimuli and induces activated and pro-inflammatory macrophages which may contribute to the development and progression of HTN. Targeting activated renal macrophages or renal GM-CSF may provide a new therapeutic option for the treatment of HTN.

Abstract 126: Nlrp3 Chromatin Availability And Isolg-adducted Histones In Salt-sensitivity Of Blood Pressure

Salt-sensitivity of blood pressure puts both normotensive and hypertensive individuals at a higher risk for cardiovascular morbidity and mortality. Isolevuglandins (IsoLGs) are increased in multiple oxidative-stress associated diseases, including salt sensitive hypertension. Scavenging of IsoLGs using 2-HOBA in a mouse model of salt-sensitive hypertension blocked NLRP3 inflammasome in dendritic cells (DCs) and abolished salt-induced hypertensive response. IsoLGs adduct to histones, specifically H4. We tested the hypothesis that elevated sodium in antigen-presenting cells leads to IsoLG-adducted H4 histones and increased chromatin availability to NLRP3 inflammasome components contributing to salt-sensitive hypertension. In vivo studies were performed on 19 hypertensive subjects enrolled in an acute inpatient protocol to assess response to salt loading (460 mmoL/24 hrs) and salt depletion (10 mmoL/24 hrs, and furosemide 40 mg x 3). Isolated PBMCs from salt sensitive subject (δ;SBP SD-SL = -12.9) and a salt resistant subject (δ;SBP SD-SL = 0.35) were analyzed by ATACseq. In vitro studies were performed on isolated CD11c+ dendritic cells treated with either normal salt (150mM) or high salt (190mM) and histone protein extraction isolated for western blot and blotted for D11, an ScFv specific to IsoLG-adducted proteins. Additional in vitro studies were performed on isolated monocytes from 11 individuals treated with either normal or high salt and submitted for RNA sequencing. RNAseq data of isolated monocytes demonstrated an overall upregulation in differentially expressed histone modification genes after high salt visualized in a heatmap and PCA plot, specifically, upregulation of gene HIST4H4 encoding the Histone H4 (25.4 ± 4.6 vs 65.5 ± 6.5, [n=11, q=1.5 x 10 -3 ]). Changes in chromatin availability inflammasome component NLRP3 were more salt responsive in DCs of the salt-sensitive subject versus the salt-resistant subject after salt depletion. Additionally, high salt increased IsoLG-histone adduction in CD11c+ DCs. Our results suggest the high sodium environment increase in IsoLG-adducts may epigenetically regulate NLRP3 inflammasome components in SSBP.

Keratin8 Deficiency Aggravates Retinal Ganglion Cell Damage Under Acute Ocular Hypertension.

Keratin 8/18 (KRT8/18), paired members of the intermediate filament family, have shown vital functions in regulating physiological activities more than supporting the mechanic strength for cells and organelles. However, the KRT8/18 presence in retinal ganglion cells (RGCs) and functions on neuroprotection in a mouse model of acute ocular hypertension (AOH) are unknown and worthy of exploration. We identified the existence of KRT8/18 in normal human and mouse retinas and primary RGCs. KRT8/18 levels were detected after AOH modeling. The adeno-associated virus (AAV) system was intravitreally used for selective KRT8 knockdown in RGCs. The histological changes, the loss and dysfunction of RGCs, and the gliosis in retinas were detected. The markers of cell apoptosis and MAPK pathways were investigated. KRT8/18 existed in all retinal layers and was highly expressed in RGCs, and they increased after AOH induction. The KRT8 knockdown in RGCs caused no histopathological changes and RGC loss in retinas without AOH modeling. However, after the KRT8 deficiency, AOH significantly promoted the loss of whole retina and inner retina thickness, the reduction, apoptosis, and dysfunction of RGCs, and the glial activation. Besides, downregulated Bcl-2 and upregulated cleaved-Caspase 3 were found in the AOH retinas with KRT8 knockdown, which may be caused by the increased phosphorylation level of MAPK pathways (JNK, p38, and ERK). The KRT8 deficiency promoted RGC apoptosis and neurodegeneration by abnormal activation of MAPK pathways in AOH retinas. Targeting KRT8 may serve as a novel treatment for saving RCGs from glaucomatous injuries.

Abstract P237: Bainiku-ekisu Attenuates Cardiac Remodeling In Angiotensin Ii-infused Mice

A fruit of the Prunus mume is a traditional food in Japan. Recently, bainiku-ekisu, an infused juice concentrate of Japanse Prunus mume , is attracting attention as a health promoting supplement. Angiotensin II (AngII) plays a central role in development of hypertension. It has been reported that bainiku-ekisu treatment attenuates the growth-promoting signaling induced by AngII in vascular smooth muscle cells. However, whether bainiku-ekisu has any effect on an animal model of hypertension remains unknown. Therefore, this study was designed to explore the potential anti-hypertensive benefit of bainiku-ekisu utilizing a mouse model of hypertension with AngII infusion. 8- to 10-week-old male C57BL6 mice were randomly divided into four groups and infused with Ang II (1 μg/kg/min) for 2 weeks via osmotic mini-pump or sham-operated, and given 1% BE containing water (BEW) or normal water (control) for 2 weeks. Blood pressure was evaluated every other day by tail cuff measurement and at 2 weeks by telemetry. Cardiac hypertrophy was assessed by heart-to-body weight ratio. After 2 weeks, mice were euthanized, and the aorta and heart collected. Extracted aortas and hearts were fixed and used for histological studies to evaluate vascular remodeling. Increase in heart-to-body weight ratios by Ang II were unaltered by BEW. Aortic medial hypertrophy was observed in control mice (n=5) after 2 weeks of Ang II infusion, which was attenuated in BEW group (96 μm vs. 71 μm, respectively, p<0.01). Control mice with angiotensin II infusion showed cardiac matrix deposition, which was attenuated in BEW group. In addition, attenuation of hypertension was observed in Ang II-infused mice with BEW treatment. We also observed attenuation of protein synthesis induced by AngII with BE incubation in cultured aortic VSMC. In conclusion, BEW prevented Ang II-induced vascular remodeling and hypertension. Potential cardiovascular health benefit to taking BE as a functional food should be further studied.

ATP-P2X7R-mediated microglia senescence aggravates retinal ganglion cell injury in chronic ocular hypertension

BackgroundDysfunction of microglia during aging affects normal neuronal function and results in the occurrence of neurodegenerative diseases. Retinal microglial senescence attributes to retinal ganglion cell (RGC) death in glaucoma. This study aims to examine the role of ATP-P2X7R in the mediation of microglia senescence and glaucoma progression.MethodsForty-eight participants were enrolled, including 24 patients with primary open-angle glaucoma (POAG) and age-related cataract (ARC) and 24 patients with ARC only. We used ARC as the inclusion criteria because of the availability of aqueous humor (AH) before phacoemulsification. AH was collected and the adenosine triphosphate (ATP) concentration was measured by ATP Assay Kit. The chronic ocular hypertension (COH) mouse model was established by microbead occlusion. Microglia were ablated by feeding PLX5622 orally. Mouse bone marrow cells (BMCs) were prepared and infused into mice through the tail vein for the restoration of microglia function. Western blotting, qPCR and ELISA were performed to analyze protein and mRNA expression in the ocular tissue, respectively. Microglial phenotype and RGC survival were assessed by immunofluorescence. The mitochondrial membrane potential was measured using a JC-1 assay kit by flow cytometry.ResultsATP concentrations in the AH were increased in older adults and patients with POAG. The expression of P2X7R was upregulated in the retinal tissues of mice with glaucoma, and functional enrichment analysis showed that P2X7R was closely related to cell aging. Through in vivo and in vitro approaches, we showed that pathological activation of ATP-P2X7R induced accelerated microglial senescence through impairing PTEN-induced kinase 1 (PINK1)-mediated mitophagy, which led to RGC damage. Additionally, we found that replacement of senescent microglia in COH model of old mice with BMCs from young mice reversed RGC damage.ConclusionATP-P2X7R induces microglia senescence by inhibiting PINK1-mediated mitophagy pathway. Specific inhibition of ATP-P2X7R may be a fundamental approach for targeted therapy of RGC injury in microglial aging-related glaucoma.

Intestinal Cckbr-specific knockout mouse as a novel model of salt-sensitive hypertension via sodium over-absorption.

To investigate the value of CCKBRfl/fl villin-Cre mice as a mouse model of salt-sensitive hypertension (SSH). In the first part, 2-month-old CCKBRfl/fl villin-Cre mice (CKO) and control CCKBRfl/fl mice (WT) were fed with normal diet (0.4% NaCl) or high salt diet (4% NaCl), separately for 6 weeks. In the rescue study, one week of hydrochlorothiazide or saline injection were treated with the CKO mice fed high salt diet. The blood pressure, biochemical indexes, and the expression of small intestinal sodium transporters (NHE3, NKCC1, eNaC) was detected. The organ injury markers (MMP2/MMP9) and the histopathological changes of kidneys were observed, whereas the changes of duodenal sodium absorption were detected by small intestinal perfusion in vivo. The CCKBRfl/fl villin-Cre mice with high salt intake exhibited high blood pressure, increased duodenal sodium absorption and urinary sodium excretion, and with renal injury. The protein expression of NHE3, NKCC1 and eNaC were also significant increase in the intestine of CKO-HS mice. Treatment with hydrochlorothiazide remarkably attenuated the elevated blood pressure by high salt absorption in the CCKBRfl/fl villin-Cre mice, but no significant histopathological changes were observed. These results support a crucial role of intestinal Cckbr deficiency on SSH development and the diuretic antihypertension effect in CCKBRfl/fl villin-Cre mice. The CCKBRfl/fl villin-Cre mice with the high salt intake may serve as a stable model of salt-sensitive hypertensive induced by sodium overloading.

Identification of ACKR4 as an immune checkpoint in pulmonary arterial hypertension.

Inflammation is recognized as a contributor in the development of pulmonary arterial hypertension (PAH), and the recruitment and functional capacity of immune cells are well-orchestrated by chemokines and their receptors. This study is aimed at identification of critical chemokines in the progression of PAH via transcriptomic analysis. Differentially expressed genes (DEGs) from lungs of PAH patients were achieved compared to controls based on Gene Expression Omnibus (GEO) database. Gene set enrichment analysis (GSEA) was applied for functional annotation and pathway enrichement. The abundance of immune cells was estimated by the xCell algorithm. Weighted correlation network analysis (WGCNA) was used to construct a gene expression network, based on which a diagnostic model was generated to determine its accuracy to distinguish PAH from control subjects. Target genes were then validated in lung of hypoxia-induce pulmonary hypertension (PH) mouse model. ACKR4 (atypical chemokine receptor 4) was downregulated in PAH lung tissues in multiple datasets. PAH relevant biological functions and pathways were enriched in patients with low-ACKR4 level according to GSEA enrichment analysis. Immuno-infiltration analysis revealed a negative correlation of activated dendritic cells, Th1 and macrophage infiltration with ACKR4 expression. Three gene modules were associated with PAH via WGCNA analysis, and a model for PAH diagnosis was generated using CXCL12, COL18A1 and TSHZ2, all of which correlated with ACKR4. The ACKR4 expression was also downregulated in lung tissues of our experimental PH mice compared to that of controls. The reduction of ACKR4 in lung tissues of human PAH based on transcriptomic data is consistent with the alteration observed in our rodent PH. The correlation with immune cell infiltration and functional annotation indicated that ACKR4 might serve as a protective immune checkpoint for PAH.

Infused juice concentrate of Japanese plum Prunus mume attenuates inflammatory vascular remodeling in a mouse model of hypertension induced by angiotensin II.

Fruit from the Prunus mume tree is a traditional food in Japan. Recently, bainiku-ekisu, an infused juice concentrate of Japanese Prunus mume, is attracting attention as a health promoting supplement. Angiotensin II (Ang II) plays a central role in development of hypertension. It has been reported that bainiku-ekisu treatment attenuates the growth-promoting signaling induced by Ang II in vascular smooth muscle cells. However, whether bainiku-ekisu has any effect on an animal model of hypertension remains unknown. Therefore, this study was designed to explore the potential anti-hypertensive benefit of bainiku-ekisu utilizing a mouse model of hypertension with Ang II infusion. Male C57BL/6 mice were infused with Ang II for 2 weeks and given 0.1% bainiku-ekisu containing water or normal water for 2 weeks with blood pressure evaluation. After 2 weeks, mice were euthanized, and the aortas were collected for evaluation of remodeling. Aortic medial hypertrophy was observed in control mice after Ang II infusion, which was attenuated in bainiku-ekisu group with Ang II infusion. Bainiku-ekisu further attenuated aortic induction of collagen producing cells and immune cell infiltration. Development of hypertension induced by Ang II was also prevented by bainiku-ekisu. Echocardiograph indicated protection of Ang II-induced cardiac hypertrophy by bainiku-ekisu. In vascular fibroblasts, bainiku-ekisu attenuated vascular cell adhesion molecule-1 induction, an endoplasmic reticulum stress marker, inositol requiring enzyme-1α phosphorylation, and enhancement in glucose consumption in response to Ang II. In conclusion, Bainiku-ekisu prevented Ang II-induced hypertension and inflammatory vascular remodeling. Potential cardiovascular health benefit to taking bainiku-ekisu should be further studied.

Intrapulmonary arterial contraction assay reveals region-specific deregulation of vasoreactivity to lung injuries.

Intrapulmonary arteries located in the proximal lung differ from those in the distal lung in size, cellular composition, and the surrounding microenvironment. However, whether these structural variations lead to region-specific regulation of vasoreactivity in homeostasis and following injury is unknown. Herein, we employ a two-step method of precision-cut lung slice (PCLS) preparation, which maintains almost intact intrapulmonary arteries, to assess contractile and relaxation responses of proximal preacinar arteries (PaAs) and distal intraacinar arteries (IaAs) in mice. We found that PaAs exhibited robust vasoconstriction in response to contractile agonists and significant nitric oxide (NO)-induced vasodilation. In comparison, IaAs were less contractile and displayed a greater relaxation response to NO. Furthermore, in a mouse model of pulmonary arterial hypertension (PAH) induced by chronic exposure to ovalbumin (OVA) allergen and hypoxia (OVA-HX), IaAs demonstrated a reduced vasocontraction despite vascular wall thickening with the emergence of new αSMA+ cells coexpressing markers of pericytes. In contrast, PaAs became hypercontractile and less responsive to NO. The reduction in relaxation of PaAs was associated with decreased expression of protein kinase G, a key component of the NO pathway, following chronic OVA-HX exposure. Taken together, the PCLS prepared using the modified preparation method enables functional evaluation of pulmonary arteries in different anatomical locations and reveals region-specific mechanisms underlying the pathophysiology of PAH in a mouse model.NEW & NOTEWORTHY Utilizing mouse precision-cut lung slices with preserved intrapulmonary vessels, we demonstrated a location-dependent structural and contractile regulation of pulmonary arteries in health and on noxious stimulations. For instance, chronic ovalbumin and hypoxic exposure increased pulmonary arterial pressure (PAH) by remodeling intraacinar arterioles to reduce vascular wall compliance while enhancing vasoconstriction in proximal preacinar arteries. These findings suggest region-specific mechanisms and therapeutic targets for pulmonary vascular diseases such as PAH.

Cardiovascular characterisation of a novel mouse model that combines hypertension and diabetes co-morbidities

Epidemiologic data suggest that the prevalence of hypertension in patients with diabetes mellitus is ∼1.5–2.0 times greater than in matched non-diabetic patients. This co-existent disease burden exacerbates cardiac and vascular injury, leading to structural and functional changes to the myocardium, impaired cardiac function and heart failure. Oxidative stress and persistent low-grade inflammation underlie both conditions, and are identified as major contributors to pathological cardiac remodelling. There is an urgent need for effective therapies that specifically target oxidative stress and inflammation to protect against cardiac remodelling. Animal models are a valuable tool for testing emerging therapeutics, however, there is a notable lack of appropriate animal models of co-morbid diabetes and hypertension. In this study, we describe a novel preclinical mouse model combining diabetes and hypertension to investigate cardiac and vascular pathology of co-morbid disease. Type 1 diabetes was induced in spontaneously hypertensive, 8-week old, male Schlager (BPH/2) mice via 5 consecutive, daily injections of streptozotocin (55 mg/kg in citrate buffer; i.p.). Non-diabetic mice received citrate buffer only. After 10 weeks of diabetes induction, cardiac function was assessed by echocardiography prior to post-mortem evaluation of cardiomyocyte hypertrophy, interstitial fibrosis and inflammation by histology, RT-PCR and flow cytometry. We focussed on the oxidative and inflammatory stress pathways that contribute to cardiovascular remodelling. In particular, we demonstrate that markers of inflammation (monocyte chemoattractant protein; MCP-1), oxidative stress (urinary 8-isoprostanes) and fibrosis (connective tissue growth factor; CTGF) are significantly increased, whilst diastolic dysfunction, as indicated by prolonged isovolumic relaxation time (IVRT), is elevated in this diabetic and hypertensive mouse model. In summary, this pre-clinical mouse model provides researchers with a tool to test therapeutic strategies unique to co-morbid diabetes and hypertension, thereby facilitating the emergence of novel therapeutics to combat the cardiovascular consequences of these debilitating co-morbidities.

Maresin-1 protects against pulmonary arterial hypertension by improving mitochondrial homeostasis through ALXR/HSP90α axis

AimsPulmonary arterial hypertension (PAH) is a progressive and lethal disease characterized by continuous proliferation of pulmonary arterial smooth muscle cell (PASMCs) and increased pulmonary vascular remodeling. Maresin-1 (MaR1) is a member of pro-resolving lipid mediators and exhibits protective effects on various inflammation-related diseases. Here we aimed to study the role of MaR1 in the development and progression of PAH and to explore the underlying mechanisms. Methods and resultsWe evaluated the effect of MaR1 treatment on PAH in both monocrotaline (MCT)-induced rat and hypoxia+SU5416 (HySu)-induced mouse models of pulmonary hypertension (PH). Plasma samples were collected from patients with PAH and rodent PH models to examine MaR1 production. Specific shRNA adenovirus or inhibitors were used to block the function of MaR1 receptors. The data showed that MaR1 significantly prevented the development and blunted the progression of PH in rodents. Blockade of the function of MaR1 receptor ALXR, but not LGR6 or RORα, with BOC-2, abolished the protective effect of MaR1 against PAH development and reduced its therapeutic potential. Mechanistically, we demonstrated that the MaR1/ALXR axis suppressed hypoxia-induced PASMCs proliferation and alleviated pulmonary vascular remodeling by inhibiting mitochondrial accumulation of heat shock protein 90α (HSP90α) and restoring mitophagy. ConclusionMaR1 protects against PAH by improving mitochondrial homeostasis through ALXR/HSP90α axis and represents a promising target for PAH prevention and treatment.

Neuroprotective Effect of a Nutritional Supplement Containing Spearmint Extract, Forskolin, Homotaurine and Group B Vitamins in a Mouse Model of Transient Ocular Hypertension.

Glaucoma is one of the most common sight-threatening eye disorders and one of the main causes of irreversible blindness worldwide. The current therapies focusing on reducing intraocular pressure (IOP) are often insufficient to prevent the progression of the disease, so the therapeutic management of glaucoma remains a challenge. The aim of this study was to evaluate the neuroprotective, IOP-lowering independent effects of a nutritional supplement containing forskolin, homotaurine, spearmint extract and vitamins of the B group in a model of acute glaucoma developed in mice. Glaucoma was induced in adult wild-type C57BL/6J mice by transient elevation of IOP. The dietary supplement, branded as Gangliomix® (125 mg/kg/day), was administered by oral gavage for 17 days and ocular hypertension was induced on the 10th day of treatment. A histological analysis of the retinas was performed and RGC survival was evaluated with fluorogold labeling and Brn3a immunostaining on wholemount and retinal sections. Expression of alpha-spectrin, caspase-3, PARP-1 and GFAP was studied with western blotting or immunofluorescence. A significant increase in RGC survival was reported in the retina of mice treated with the dietary supplement as compared to vehicle-treated animals. The observed neuroprotection was associated with a calpain activity decrease, reduction in caspase-3 and PARP-1 activation, and prevention of GFAP upregulation. These effects were independent from the hypotensive effects of the supplement. Altogether, our data suggest that the dietary supplementation with forskolin, homotaurine, spearmint extract and vitamins of the B group supports RGC survival and may offer beneficial effects in glaucoma patients in combination with the currently used IOP-lowering therapy.

Regulation of renal function by the peroxisome proliferator-activated receptor-alpha: A novel target for treating hypertension

Approximately 37 million people in the U.S. have chronic kidney disease, which is a major risk factor for cardiovascular and end stage renal diseases. We have identified a nuclear receptor, peroxisome proliferator-activated receptor – alpha (PPAR-α), that plays a major role in regulating renal glomerular filtration rate in an experimental model of hypertension. Targeting PPAR-α signaling provides novel therapeutic strategies for reducing hypertension associated chronic kidney disease. PPAR-α knockout (KO) mice exhibit increased renal inflammation and blood pressure response to Angiotensin II (Ang II). In this study, we investigated the role of PPAR-α in renal function in a mouse model of hypertension. Male 4-month-old wild type (WT) and PPAR-α KO mice were instrumented with radio transmitters by artery canulation (Data Science International). This method minimizes stress and artifacts by avoiding the use of tethering, restraining, or anesthetizing the mice during data sampling. After recovery from surgery, we continuously measured mean arterial pressure (MAP) via radio telemetry in conscious ambulatory mice. After baseline MAP was established, vehicle (Veh; saline) or Ang II were infused using an osmotic minipump at a slow pressor dose (400 ng/kg/min) for 12 days. On day 12, we injected an intravenous bolus of fluorescin-sinistrin (3.74 ml/g body weight) and collected 8 blood samples (20 μl/sample) during a 75-minute period to enable calculation of the glomerular filtration rate (GFR) using [GFR = I/(A/a + B/b]. Similar to our prior observations, no significant (ns) differences in baseline MAP were observed between WT and PPAR-α KO mice [(mmHg): WT (n=6), 111±20 vs. PPAR-α KO (n=6), 113 ± 10; ns] whereas after 12 days of the slow pressor effect of Ang II, MAP was increased in both strains [(mmHg): WT (n=8), 138 ± 11# vs. PPAR-α KO (n=8), 156 ± 16#; #p<0.05 vs. basal, same strain]; however, the increase in MAP was greater in the PPAR-α KO (43 mmHg) compared to WT (27 mmHg) mice. The absence of PPAR-α reduced GFR by 24% under basal conditions [(μl/min): WT (n=3), 346 ± 20 vs. PPAR-α KO (n=3), 275 ± 27*, *p<0.05 vs WT]. After 12 days of Ang II infusion, GFR was reduced in WT mice by 27% and did not further reduce GFR in PPAR-α KO mice [(μl/min): WT (n=6), 251 ± 11# vs. PPAR-α KO (n=6), 277 ± 61; #p<0.05 vs. basal, same strain]. PPAR-α protects mice from worsening hypertension and is critical to preserving GFR during normotensive conditions. Ongoing studies are further investigating how PPAR-α regulates renal function. These finding suggest therapeutics designed to increase PPAR-α activity could have clinical benefit in chronic kidney disease. Howard University Research Centers at Minority Institutions, Georgetown-Howard Universities Center for Clinical and Translational Science, Howard University Aging Grant, K01HL092593 This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.