Angiotensin II Treatment Research Articles

Hypertension-induced heart failure disrupts cardiac sympathetic innervation.

About 26 million people worldwide live with heart failure (HF), and hypertension is the primary cause in 25% of these cases. Autonomic dysfunction and sympathetic hyperactivity accompany cardiovascular diseases, including HF. However, changes in cardiac sympathetic innervation in HF are not well understood. We hypothesized that cardiac sympathetic innervation is disrupted in hypertension-induced HF. Male and female C57BL6/J mice were infused with Angiotensin II (AngII) for 4 weeks to generate hypertension leading to HF; controls were infused with saline. AngII-treated mice displayed HF phenotype including reduced cardiac function, hypertrophy, and fibrosis. AngII-treated mice also had significantly reduced sympathetic nerve density in the left ventricle, intraventricular septum, and right ventricle. In the left ventricle, the subepicardium remained normally innervated, while the subendocardium was almost devoid of sympathetic nerves. Loss of sympathetic fibers led to loss of norepinephrine content in the left ventricle. Several potential triggers for axon degeneration were tested and ruled out. AngII-treated mice had increased premature ventricular contractions after isoproterenol and caffeine injection. Although HF can induce a cholinergic phenotype and neuronal hypertrophy in stellate ganglia, AngII treatment did not induce a cholinergic phenotype or activation of trophic factors in this study. Cardiac neurons in the left stellate ganglion were significantly smaller in AngII-treated mice, while neurons in the right stellate were unchanged. Our findings show that AngII-induced HF disrupts sympathetic innervation, particularly in the left ventricle. Further investigations are imperative to unveil the mechanisms of denervation in HF and to develop neuromodulatory therapies for patients with autonomic imbalance.

Angiotensin II as a linking factor in cardiovascular disease enhanced cancer growth

Abstract Introduction Several publications have demonstrated that cardiovascular diseases (CVD) promote tumor growth in mouse models of breast, lung, and colon cancer, suggesting a causal relationship between both diseases. Cancer and CVD share several risk factors and pathophysiological mechanisms, including inflammation, oxidative stress, neurohormonal activation, and immune system dysfunction that combined could explain why patients with CVD are prone to develop cancer. Recent reports also suggest a link between hypertension and lung cancer, and between cardiac hypertrophy and breast cancer. Angiotensin II (ANGII) is a hormone involved in blood pressure regulation, vascular hemostasis and hypertrophy development. Moreover, ANGII is cancerogenic by increasing proliferation of several cancer cell types. Hence, ANGII could be a link in CVD-induced enhancement of cancer growth. Here, we investigated whether ANGII treatment, and subsequent hypertension and cardiac hypertrophy, could promote cancer growth and metastasis. Methods ANGII (2000ng.kg-1.min-1, 4w) was administered to 10w old C57B6/J mice using subcutaneous osmotic minipumps. After 3w, 5*10^5 Lewis lung carcinoma (LLC) cells or 2*10^5 MC38 colon cancer cells were injected onto the right flank of the mice. Tumor growth was monitored over 21d using a digital caliper. Tumor volume was calculated using the following formula: Length × width² × 0.5 On day 22, mice were sacrificed for further post-mortem analysis. Cardiac echography was performed after 3w of ANGII treatment and before sacrifice to assess cardiac function (data not shown). To assess the formation of cardiac fibrosis trichrome staining’s were performed. In addition, the effect of ANGII on LLC metastasis was investigated. Mice were treated with ANGII as described above. LLC cells, 2*10^6, were injected intravenously (i.v.). Mice were sacrificed 21d after cell injection. Metastatic growth was visualized and quantified by organ weighing and H&E staining. Results LLC tumor growth was significantly increased by ANGII treatment. In addition, post-mortem tumor weight was increased (0.62 ± 0.05 g control vs. 1.17 ± 0.24 g ANGII). Furthermore, we found lung weight increased in the ANGII-treated group injected i.v. with LLC cells, indicating an increase in metastatic growth (0.41 ± 0.08 g control vs. 0.57 ± 0.04 g ANGII). Interestingly, MC38 tumor growth decreased in the ANGII treated group as portrayed by the tumor weight (0.42 ± 0.06 g control vs. 0.19 ± 0.05 g ANGII). Figure 1 shows the tumor growth over time and tumor volume, weight post-mortem. Conclusion Our data suggest a role for ANGII in CVD-enhanced cancer growth, but also that this effect is not universal, illustrated by a differential effect of ANGII on distinct cancer types. Although further investigation is required to unravel the role of ANGII, screening cancer patients with CVD comorbidities for ANGII involvement might be warranted.

Endothelial NRG-1 knock-out causes left ventricle dilation in the presence and absence of angiotensin II administration

Abstract Introduction Neuregulin-1 (NRG-1) is a growth factor derived from endothelial cells and belongs to the epidermal growth factor (EGF) family. While NRG-1 is expressed by various cell types, including epithelial cells, glial cells, neurons, and myocytes, the primary source of NRG-1 is considered to be the endothelium. NRG-1 exerts its effects through the EGF receptors ERBB2, ERBB3, and ERBB4. Upon ligand binding, these receptors dimerize and become phosphorylated, leading to downstream signaling that impacts tissue proliferation, differentiation, and survival. The biological role of NRG-1 is intricate due to its isoforms and differential expression across distinct tissue types. In our study, we investigated the impact of endothelial NRG-1 knock-out (KO) on the heart, both in the presence and absence of angiotensin II (ANGII) treatment. ANGII serves as a trigger for NRG-1 upregulation. Methods C57BL/6N-VE-cad-cre-ERT2 mice were crossbred with C57BL/6N-NRG F/F mice, enabling endothelial-specific NRG-1 knockout (KO) using a tamoxifen-inducible promoter. At eight weeks of age, C57BL/6N-VE-cad-cre-ERT2-NRG F/F mice received tamoxifen (1 mg) or vehicle injections for nine consecutive days intraperitoneally (i.p.) to induce the KO. Two days after injection, osmotic minipumps were subcutaneously implanted, releasing a stable dose of angiotensin II (ANGII) at a rate of 400 ng·kg·min⁻¹ for a four-week period. Sham operations served as controls (SHAM). In the final week of treatment, high-frequency ultrasound imaging was performed to assess cardiac function and dimensions. After four weeks of ANGII treatment, mice were sacrificed, and organs were weighed and harvested for further molecular analysis. Results High-frequency ultrasound revealed left ventricle dilation in the absence of endothelial NRG-1, both in ANGII-treated and SHAM-treated mice. This was demonstrated by a significant increase in systolic and diastolic left ventricle internal diameter (LVID) in the endothelial NRG-1 KO groups compared to their respective controls. Additionally, systolic and diastolic LV volume (LV-vol) was significantly increased in the endothelial NRG-1 KO groups, regardless of ANGII treatment. Notably, there was no difference in LVID and LV-vol between SHAM and ANGII-treated groups in the presence of endothelial NRG-1, indicating that LV dilation is primarily due to the absence of endothelial NRG-1 rather than ANGII. Furthermore, heart weights of the endothelial NRG-1 KO mice were significantly increased compared to the control groups, as shown in Figure 1. Conclusion Our study demonstrates that endothelial NRG-1 KO leads to LV dilation, suggesting a crucial role for endothelial-secreted NRG-1 in maintaining normal LV function in adulthood. Further research is needed to fully understand the precise role of NRG-1 in LV homeostasis.

Abstract Mo019: Angiotensin II Links Cardiovascular Disease With Enhanced Cancer Growth

Introduction: It has been demonstrated that CVD promote tumor growth in mouse models of breast, lung, and colon cancer, suggesting a causal relationship between both diseases. Cancer and CVD share several risk factors and pathophysiological mechanisms, e.g. inflammation and oxidative stress, that could explain why patients with CVD are prone to develop cancer. Recent reports also suggest a link between hypertension, hypertrophy and lung cancer. Angiotensin II (ANGII) is a hormone involved in blood pressure regulation, vascular hemostasis and hypertrophy development. Moreover, ANGII is cancerogenic by increasing proliferation of several cancer cell types. Hence, ANGII could be a link in CVD-induced enhancement of cancer growth. Hypothesis: We hypothesized that ANGII treatment, and subsequent hypertension and cardiac hypertrophy, could promote cancer growth. Methods: ANGII (2000 ng.kg -1 .min -1 ; 4w) was administered to 10w old C57B6/J mice using osmotic minipumps. After 4w, pumps were removed and 5*10 5 Lewis lung carcinoma (LLC) cells injected onto the right flank. To study the direct impact of the ANGII peptide on the cancer growth mice were treated with ANGII as mentioned above and LLC cells were injected after 3w to allow concomitant ANGII treatment and cancer growth. A similar experiment was performed by injecting 2*10 5 MC38 colon cancer cells. Tumor growth was monitored for 21d after which the mice were sacrificed. Results are expressed as mean ± SEM. Results: ANGII treatment concomitantly with cancer growth resulted in significantly larger tumors as shown by post-mortem tumor volume (881 ± 111 control vs. 1467 ± 154 mm^3 ANGII, P=0.013). In contrast, no increased tumor growth was observed when ANGII was administered non-concomitant with cancer growth. Interestingly, MC38 tumor growth significantly decreased in the ANGII treated group as portrayed by tumor volume (462 ± 65 control vs. 227 ± 81 mm^3 ANGII). Conclusion: Our data suggest a role for ANGII in CVD-enhanced cancer growth, but also that this effect is not universal, illustrated by a differential effect of ANGII on distinct cancer types. Although further investigation is required to unravel the role of ANGII, screening cancer patients with CVD comorbidities for ANGII involvement might be warranted.

Disruption of the interaction between caveolae and Piezo1 promotes pressure overload-induced cardiac remodeling

Piezo1 channels are activated by mechanical stress and play a significant role in cardiac hypertrophy and fibrosis. However, the molecular mechanisms underlying Piezo1 activation on the cell membrane following pressure overload remain unclear. Caveolae are known to mitigate mechanical forces and regulate Piezo1 function. Therefore, this study aimed to investigate the interaction between caveolae and Piezo1 in the development of pressure overload-induced cardiac remodeling. We observed reduced colocalization between Piezo1 and Caveolin-3 in hypertrophic cardiomyocytes following abdominal aortic constriction and Angiotensin-II treatment, accompanied by increased Piezo1 function and expression. Furthermore, enhanced Piezo1 function was also noted upon caveolae disruption using methyl-beta-cyclodextrin (mβCD). Thus, our findings suggested that pressure overload led to Piezo1 translocation from caveolae, thereby augmenting its function and expression, which may contribute to cardiac remodeling.

KLF4 in smooth muscle cell-derived progenitor cells is essential for angiotensin II-induced cardiac inflammation and fibrosis.

Cardiac fibrosis is defined by the excessive accumulation of extracellular matrix (ECM) material resulting in cardiac tissue scarring and dysfunction. While it is commonly accepted that myofibroblasts are the major contributors to ECM deposition in cardiac fibrosis, their origin remains debated. By combining lineage tracing and RNA sequencing, our group made the paradigm-shifting discovery that a subpopulation of resident vascular stem cells residing within the aortic, carotid artery, and femoral aartery adventitia (termed AdvSca1-SM cells) originate from mature vascular smooth muscle cells (SMCs) through an in situ reprogramming process. SMC-to-AdvSca1-SM reprogramming and AdvSca1-SM cell maintenance is dependent on induction and activity of the transcription factor, KLF4. However, the molecular mechanism whereby KLF4 regulates AdvSca1-SM phenotype remains unclear. In the current study, leveraging a highly specific AdvSca1-SM cell reporter system, single-cell RNA-sequencing (scRNA-seq), and spatial transcriptomic approaches, we demonstrate the profibrotic differentiation trajectory of coronary artery-associated AdvSca1-SM cells in the setting of Angiotensin II (AngII)-induced cardiac fibrosis. Differentiation was characterized by loss of stemness-related genes, including Klf4 , but gain of expression of a profibrotic phenotype. Importantly, these changes were recapitulated in human cardiac hypertrophic tissue, supporting the translational significance of profibrotic transition of AdvSca1-SM-like cells in human cardiomyopathy. Surprisingly and paradoxically, AdvSca1-SM-specific genetic knockout of Klf4 prior to AngII treatment protected against cardiac inflammation and fibrosis, indicating that Klf4 is essential for the profibrotic response of AdvSca1-SM cells. Overall, our data reveal the contribution of AdvSca1-SM cells to myofibroblasts in the setting of AngII-induced cardiac fibrosis. KLF4 not only maintains the stemness of AdvSca1-SM cells, but also orchestrates their response to profibrotic stimuli, and may serve as a therapeutic target in cardiac fibrosis.

Angiotensin II-Induced Heart Failure Disrupts Cardiac Sympathetic Innervation

About 26 million people worldwide live with heart failure (HF), and 25% have hypertension (HTN) as the primary cause. These patients have a high risk for cardiac arrhythmias and sudden cardiac death. Autonomic dysfunction and sympathetic hyperactivity accompany these diseases, and sympathetic activation is crucial for triggering fatal arrhythmias. However, changes in cardiac sympathetic innervation in HF are not well understood. We hypothesized that cardiac sympathetic innervation is disrupted in a mouse model of Angiotensin II (AngII)-induced HF. To test this hypothesis, C57BL6 male (N=6, 10) and female (N=6, 10) mice 12-20 weeks old were implanted with minipumps releasing either AngII (712ng/min/Kg, N=6-10) or saline (N=6-10) for 4 weeks. HTN and cardiac function were measured with tail cuff quantification and echocardiography, respectively. Fibrosis was assessed using Trichrome staining. Cardiac sympathetic innervation was quantified using immunohistochemistry for Tyrosine Hydroxylase (TH). Cardiac norepinephrine (NE) was measured by High-Performance Liquid Chromatography with electrochemical detection. Values were compared with a Student’s unpaired t-test unless noted, data were represented as mean ± SD, and significance was defined as p<0.05. AngII-treated mice had significantly increased mean arterial pressure (Saline 91.0±15.6 vs. AngII 152.9±22.3 mmHg, p<0.05, N=6-7) 7 days after minipump implantation. Four weeks of AngII treatment led to HF, as indicated by reduced ejection fraction (Saline 54.8±3.8 vs. AngII 43.1±8.2 %, p<0.05, N=10) and fractional shortening (Saline 14.3±2.6 vs. AngII 10.6±3.3 %, p<0.05, N=10). Cardiac hypertrophy was also observed in AngII-treated mice and was reflected by increased heart weight to body weight ratio (Saline 5.2±0.2 vs. AngII 7.7±1.0, p<0.05, N=6). Fibrosis was detected in the intraventricular septum (IVS) and left ventricle (LV) of AngII-treated mice. TH+ fibers in the LV and IVS were decreased significantly (LV: Saline 0.6±0.2 vs. AngII 0.4±0.1 %; IVS: Saline 0.3±0.1 vs. AngII 0.09±0.04 %, p<0.05, N=6), while no differences were detected in the right ventricle (Saline 1.1±0.2 vs. AngII 0.8±0.3 %, P>0.05, N=6). In addition, the subepicardium-subendocardium (EPI-ENDO) gradient was altered (EPI: Saline 0.9±0.2 vs. AngII 0.8±0.3 %, P>0.05; ENDO: Saline 0.3±0.05 vs. AngII 0.1±0.1 %, p<0.05; EPI:ENDO ratio: Saline 2.7±0.7 vs. AngII 15.7±16.8, p<0.05, Mann-Whitney test, N=6). A significant decrease in NE content accompanied the loss of TH+ fibers in the LV (LV: Saline 3.9±0.4 vs. AngII 1.6±0.2 pmol/mg, p<0.05; RV: Saline 4.4±1.0 vs. AngII 3.8±0.8 pmol/mg, P>0.05, N=4-6). Our data demonstrate that AngII-induced HF disrupts cardiac sympathetic nerve density, distribution, and NE content, confirming our initial hypothesis. This evidence contrasts with sympathetic hyperactivity seen in cardiac diseases. Future studies will assess the overall cardiac innervation and, determine whether the loss of TH+ fibers is due to a reduced sympathetic innervation after cardiac remodeling or the cholinergic transdifferentiation of TH+ fibers. A better understanding of the cardiac sympathetic nervous system in HF is imperative to deciphering the mechanistic changes to cardiac innervation in disease, and may help to develop and implement novel and precise neuromodulatory therapies for patients with autonomic imbalance. Supported by NIH R01 HL146833 to Dr. Habecker. This is the full abstract presented at the American Physiology Summit 2024 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.

Abstract 2149: Smooth Muscle-derived Resident Vascular Adventitial Progenitor Cell Contribution To Cardiac Fibrosis And Inflammation Is Dependent On KLF4

Cardiac fibrosis is defined by the excessive accumulation of extracellular matrix (ECM) material resulting in cardiac tissue scarring and dysfunction. While it is commonly accepted that myofibroblasts are the major contributors to ECM deposition in cardiac fibrosis, their origin remains debated. By combining lineage tracing and RNA sequencing, our group made the paradigm-shifting discovery that a subpopulation of resident vascular stem cells residing within the vascular adventitia (termed AdvSca1-SM cells) originate from mature vascular smooth muscle cells (SMCs) through an in situ reprogramming process. SMC-to-AdvSca1-SM reprogramming and AdvSca1-SM cell maintenance is dependent on induction and activity of the transcription factor, KLF4. In contrast, loss of Klf4 expression promotes the transition of AdvSca1-SM cells to a myofibroblast phenotype in vascular injury and remodeling. However, the molecular mechanism whereby KLF4 regulates AdvSca1-SM phenotype remains unclear. Our recent preliminary data confirm the existence of perivascular cardiac AdvSca1-SM cells. Leveraging a highly specific AdvSca1-SM cell reporter system, single-cell RNA-sequencing (scRNA-seq), and spatial transcriptomic approaches, we demonstrate the profibrotic differentiation trajectory of AdvSca1-SM cells in the setting of Angiotensin II (AngII)-induced cardiac fibrosis. Differentiation is characterized by loss of stemness-related genes, including Klf4 , but gain of expression of a profibrotic phenotype. Importantly, these changes are recapitulated in human cardiac hypertrophic tissue, supporting the translational significance of profibrotic transition of AdvSca1-SM-like cells in human cardiomyopathy. Very surprisingly and paradoxically, AdvSca1-SM-specific genetic knockout of Klf4 prior to AngII treatment protects against cardiac inflammation and fibrosis, indicating that Klf4 is essential for the profibrotic response of AdvSca1-SM cells. Overall, our data reveal the contribution of AdvSca1-SM cells to myofibroblasts in the setting of cardiac fibrosis. KLF4 not only maintains the stemness of AdvSca1-SM cells, but also orchestrates their response to profibrotic stimuli, and may serve as a therapeutic target in cardiac fibrosis.

ACE inhibitors and angiotensin receptor blockers differentially alter the response to angiotensin II treatment in vasodilatory shock

BackgroundAngiotensin-converting enzyme inhibitor (ACEi) and angiotensin receptor blockers (ARB) medications are widely prescribed. We sought to assess how pre-admission use of these medications might impact the response to angiotensin-II treatment during vasodilatory shock.MethodsIn a post-hoc subgroup analysis of the randomized, placebo-controlled, Angiotensin Therapy for High Output Shock (ATHOS-3) trial, we compared patients with chronic angiotensin-converting enzyme inhibitor (ACEi) use, and patients with angiotensin receptor blocker (ARB) use, to patients without exposure to either ACEi or ARB. The primary outcome was mean arterial pressure after 1-h of treatment. Additional clinical outcomes included mean arterial pressure and norepinephrine equivalent dose requirements over time, and study-drug dose over time. Biological outcomes included baseline RAS biomarkers (renin, angiotensin-I, angiotensin-II, and angiotensin-I/angiotensin-II ratio), and the change in renin from 0 to 3 h.ResultsWe included n = 321 patients, of whom, 270 were ACEi and ARB-unexposed, 29 were ACEi-exposed and 22 ARB-exposed. In ACEi/ARB-unexposed patients, angiotensin-treated patients, compared to placebo, had higher hour-1 mean arterial pressure (9.1 mmHg [95% CI 7.6–10.1], p < 0.0001), lower norepinephrine equivalent dose over 48-h (p = 0.0037), and lower study-drug dose over 48-h (p < 0.0001). ACEi-exposed patients treated with angiotensin-II showed similarly higher hour-1 mean arterial pressure compared to ACEi/ARB-unexposed (difference in treatment-effect: − 2.2 mmHg [95% CI − 7.0–2.6], pinteraction = 0.38), but a greater reduction in norepinephrine equivalent dose (pinteraction = 0.0031) and study-drug dose (pinteraction < 0.0001) over 48-h. In contrast, ARB-exposed patients showed an attenuated effect of angiotensin-II on hour-1 mean arterial pressure versus ACEi/ARB-unexposed (difference in treatment-effect: − 6.0 mmHg [95% CI − 11.5 to − 0.6], pinteraction = 0.0299), norepinephrine equivalent dose (pinteraction < 0.0001), and study-drug dose (pinteraction = 0.0008). Baseline renin levels and angiotensin-I/angiotensin-II ratios were highest in ACEi-exposed patients. Finally, angiotensin-II treatment reduced hour-3 renin in ACEi/ARB-unexposed and ACEi-exposed patients but not in ARB-exposed patients.ConclusionsIn vasodilatory shock patients, the cardiovascular and biological RAS response to angiotensin-II differed based upon prior exposure to ACEi and ARB medications. ACEi-exposure was associated with increased angiotensin II responsiveness, whereas ARB-exposure was associated with decreased responsiveness. These findings have clinical implications for patient selection and dosage of angiotensin II in vasodilatory shock.Trial Registration ClinicalTrials.Gov Identifier: NCT 02338843 (Registered January 14th 2015).

544 Interleukin-6 protects renal dysfunction in mouse models of hypertension and salt-sensitive hypertension

OBJECTIVES/GOALS: We are investigating the role of IL-6 in regulating renal function by measuring mean arterial pressure (MAP), renal plasma flow (RPF) and glomerular filtration rate (GFR) in wild type (WT) and IL-6-knockout (KO) mice in established mouse models of angiotensin II (AII)-dependent- hypertension and -salt-sensitive hypertension. METHODS/STUDY POPULATION: Twelve-week-old male WT and KO mice on the C57BL6 background strain were infused with vehicle (V; saline) or angiotensin II (AII; 200 ng/kg/min) for 12-14 days. Half of the AII-treatment groups were maintained on a high salt (HS; 6% NaCl) diet for the duration of the experiment, while the other half of the AII treatment groups and both vehicle groups were fed normal rat chow. MAP was continuously measured by a fluid filled catheter in conscious mice for the duration of the experiment. RPF and GFR were measured on days 12-14 in anesthetized mice by the para-aminohippurate, and fluorescein isothiocynate-Inulin techniques, respectively. All data were analyzed by 2-way ANOVA; *p&lt;0.05 vs. WT, same treatment; #p&lt;0.05 vs.V, same genotype; ^p&lt;0.05, AII vs. AII+HS, same genotype. RESULTS/ANTICIPATED RESULTS: MAP was 31% lower in KO vs WT mice. AII increased MAP (1.2-fold) in WT but not KO mice. HS diet magnified AII-induced increases in MAP in WT and moderately increased MAP in AII-KO mice: [MAP (mmHg): WT+V, 130±7.0; KO+V, 91.0+4.0*; WT+AII, 153±5.0#; KO+AII, 83.0±4.0*; WT+AII+HS, 150±11#; KO+AII+HS, 93.0±4.0#]. AII infusion reduced RPF in the KO but not WT mice. Addition of HS reduced RPF in WT and exacerbated AII-induced reductions in RPF in KO mice [RPF (ml/min/g): WT+V, 1.82±0.23; KO+V, 1.91+0.40; WT+AII, 3.16±0.75#; KO+AII, 1.65±0.42*; WT+AII+HS, 1.10±0.31#^; KO+AII+HS, 1.13±0.XX#^]. The HS diet reduced GFR in AII-infused KO but not WT mice [GFR (µl/min/g): WT+V, 756±XX; KO+V, 788±XX; WT+AII, 1010±63*#; KO+AII, 756±23*; WT+AII+HS, 1100±150#; KO+AII+HS: 540±210*#^]. DISCUSSION/SIGNIFICANCE: The absence of IL-6 in male mice attenuated AII- and/or AII+HS-induced increases in MAP; however, it exacerbated HS-induced reductions in RPF and GFR. These findings suggest inhibiting IL-6 has therapeutic potential as an antihypertensive but not as a renal protective agent in hypertension and salt-sensitive hypertension disease states.

Angiotensin II treatment is associated with improved oxygenation in ARDS patients with refractory vasodilatory shock

BackgroundThe physiological effects of renin-angiotensin system modulation in acute respiratory distress syndrome (ARDS) remain controversial and have not been investigated in randomized trials. We sought to determine whether angiotensin-II treatment is associated with improved oxygenation in shock-associated ARDS.MethodsPost-hoc subgroup analysis of the Angiotensin Therapy for High Output Shock (ATHOS-3) trial. We studied patients who met modified Berlin ARDS criteria at enrollment. The primary outcome was PaO2/FiO2-ratio (P:F) at 48-h adjusted for baseline P:F. Secondary outcomes included oxygenation index, ventilatory ratio, PEEP, minute-ventilation, hemodynamic measures, patients alive and ventilator-free by day-7, and mortality.ResultsOf 81 ARDS patients, 34 (42%) and 47 (58%) were randomized to angiotensin-II or placebo, respectively. In angiotensin-II patients, mean P:F increased from 155 mmHg (SD: 69) at baseline to 265 mmHg (SD: 160) at hour-48 compared with no change with placebo (148 mmHg (SD: 63) at baseline versus 164 mmHg (SD: 74) at hour-48)(baseline-adjusted difference: + 98.4 mmHg [95%CI 35.2–161.5], p = 0.0028). Similarly, oxygenation index decreased by − 6.0 cmH2O/mmHg at hour-48 with angiotensin-II versus − 0.4 cmH2O/mmHg with placebo (baseline-adjusted difference: -4.8 cmH2O/mmHg, [95%CI − 8.6 to − 1.1], p = 0.0273). There was no difference in PEEP, minute ventilation, or ventilatory ratio. Twenty-two (64.7%) angiotensin-II patients had sustained hemodynamic response to treatment at hour-3 versus 17 (36.2%) placebo patients (absolute risk-difference: 28.5% [95%CI 6.5–47.0%], p = 0.0120). At day-7, 7/34 (20.6%) angiotensin-II patients were alive and ventilator-free versus 5/47(10.6%) placebo patients. Day-28 mortality was 55.9% in the angiotensin-II group versus 68.1% in the placebo group.ConclusionsIn post-hoc analysis of the ATHOS-3 trial, angiotensin-II was associated with improved oxygenation versus placebo among patients with ARDS and catecholamine-refractory vasodilatory shock. These findings provide a physiologic rationale for trials of angiotensin-II as treatment for ARDS with vasodilatory shock.Trial Registration: ClinicalTrials.Gov Identifier: NCT02338843 (Registered January 14th 2015).

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

Epigenome-wide DNA methylation profiling in comparison between pathological and physiological hypertrophy of human cardiomyocytes.

Background: Physiological and pathological stimuli result in distinct forms of cardiac hypertrophy, but the molecular regulation comparing the two, especially at the DNA methylation level, is not well understood. Methods: We conducted an in vitro study using human cardiomyocytes exposed to angiotensin II (AngII) and insulin-like growth factor 1 (IGF-1) to mimic pathologically and physiologically hypertrophic heart models, respectively. Whole genome DNA methylation patterns were profiled by the Infinium human MethylationEPIC platform with >850K DNA methylation loci. Two external datasets were used for comparisons and qRT-PCR was performed for examining expression of associated genes of those identified DNA methylation loci. Results: We detected 194 loci that are significantly differentially methylated after AngII treatment, and 206 significant loci after IGF-1 treatment. Mapping the significant loci to genes, we identified 158 genes corresponding to AngII treatment and 175 genes to IGF-1 treatment. Using the gene-set enrichment analysis, the PI3K-Akt signaling pathway was identified to be significantly enriched for both AngII and IGF-1 treatment. The Hippo signaling pathway was enriched after IGF-1 treatment, but not for AngII treatment. CDK6 and RPTOR are components of the PI3K-Akt pathway but have different DNA methylation patterns in response to AngII and IGF-1. qRT-PCR confirmed the different gene expressions of CDK6 and PRTOR. Conclusion: Our study is pioneering in profiling epigenome DNA methylation changes in adult human cardiomyocytes under distinct stress conditions: pathological (AngII) and physiological (IGF-1). The identified DNA methylation loci, genes, and pathways might have the potential to distinguish between pathological and physiological cardiac hypertrophy.

Micro-lightguide spectrophotometry assessment of hepatic and intestinal microcirculation in endotoxemic rats during intravenous treatment with angiotensin II

IntroductionDuring septic shock, impairment of microcirculation leads to enhanced permeability of intestinal mucosa triggered by generalized vasodilation and capillary leak. Intravenous angiotensin II (AT-II) has been approved for the treatment of septic shock; however, no in-vivo data exist on the influence of AT-II on hepatic and intestinal microcirculation. Material and methodsSixty male Lewis rats were randomly assigned to six study groups (each n = 10): sham, lipopolysaccharide-induced septic shock, therapy with low- or high-dose AT-II (50 or 100 ng/kg/min, respectively), and septic shock treated with low- or high-dose AT-II. After median laparotomy, hepatic and intestinal microcirculation measures derived from micro-lightguide spectrophotometry were assessed for 3 h and included oxygen saturation (SO2), relative blood flow (relBF) and relative hemoglobin level (relHb). Hemodynamic measurements were performed using a left ventricular conductance catheter, and blood samples were taken hourly to analyze blood gasses and systemic cytokines. ResultsAT-II increased mean arterial pressure in a dose-dependent manner in both septic and non-septic animals (p < 0.001). Lower hepatic and intestinal SO2 (both p < 0.001) were measured in animals without endotoxemia who received high-dose AT-II treatment, however, significantly impaired cardiac output was also reported in this group (p < 0.001). In endotoxemic rats, hepatic relBF and relHb were comparable among the treatment groups; however, hepatic SO2 was reduced during low- and high-dose AT-II treatment (p < 0.001). In contrast, intestinal SO2 remained unchanged despite treatment with AT-II. Intestinal relBF (p = 0.028) and interleukin (IL)-10 plasma levels (p < 0.001) were significantly elevated during treatment with high-dose AT-II compared with low-dose AT-II. ConclusionsA dose-dependent decrease of hepatic and intestinal microcirculation during therapy with AT-II in non-septic rats was observed, which might have been influenced by a corresponding reduction in cardiac output due to elevated afterload. While hepatic microcirculation was reduced during endotoxemia, no evidence for a reduction in intestinal microcirculation facilitated by AT-II was found. In contrast, both intestinal relBF and anti-inflammatory IL-10 levels were increased during high-dose AT-II treatment.

CEBPD REGULATES OXIDATIVE STRESS AND INFLAMMATORY RESPONSES IN HYPERTENSIVE CARDIAC REMODELING.

Hypertension seems to inevitably cause cardiac remodeling, increasing the mortality of patients. This study aimed to explore the molecular mechanism of CCAAT/enhancer-binding protein delta (CEBPD)-mediated oxidative stress and inflammation in hypertensive cardiac remodeling. The hypertensive murine model was established through angiotensin-II injection, and hypertensive mice underwent overexpressed CEBPD vector injection, cardiac function evaluation, and observation of histological changes. The cell model was established by angiotensin-II treatment and transfected with overexpressed CEBPD vector. Cell viability and surface area and oxidative stress (reactive oxygen species/superoxide dismutase/lactate dehydrogenase/malondialdehyde) were assessed, and inflammatory factors (TNF-α/IL-1β/IL-6/IL-10) were determined both in vivo and in vitro . The levels of CEBPD, miR-96-5p, inositol 1,4,5-trisphosphate receptor 1 (IP3R), natriuretic peptide B, and natriuretic peptide A, collagen I, and collagen III in tissues and cells were determined. The binding relationships of CEBPD/miR-96-5p/IP3R 3' untranslated region were validated. CEBPD was reduced in cardiac tissue of hypertensive mice, and CEBPD upregulation improved cardiac function and attenuated fibrosis and hypertrophy, along with reductions of reactive oxygen species/lactate dehydrogenase/malondialdehyde/TNF-α/IL-1β/IL-6 and increases in superoxide dismutase/IL-10. CEBPD enriched on the miR-96-5p promoter to promote miR-96-5p expression, whereas CEBPD and miR-96-5p negatively regulated IP3R. miR-96-5p silencing/IP3R overexpression reversed the alleviative role of CEBPD overexpression in hypertensive mice. In summary, CEBPD promoted miR-96-5p to negatively regulate IP3R expression to inhibit oxidative stress and inflammation, thereby alleviating hypertensive cardiac remodeling.

The CXCR4-AT1 axis plays a vital role in glomerular injury via mediating the crosstalk between podocyte and mesangial cell

Glomeruli stand at the center of nephrons to accomplish filtration and albumin interception.Podocytes and mesangial cells are the major constituents in the glomeruli. However, their interdependency in glomerular injury has rarely been reported. Herein, we investigated the role of C-X-C chemokine receptor type 4 (CXCR4) in mediating the crosstalk between podocytes and mesangial cells. We found CXCR4 and angiotensin II (AngII) increased primarily in injured podocytes. However, type-1 receptor of angiotensin II (AT1) and stromal cell-derived factor 1α (SDF-1α), a ligand of CXCR4, were evidently upregulated in mesangial cells following the progression of podocyte injury. Ectopic expression of CXCR4 in 5/6 nephrectomy mice increased the decline of renal function and glomerular injury, accelerated podocyte injury and mesangial cell activation, and initiated CXCR4-AT1 axis signals. Additionally, treatment with losartan, an AT1 blocker, interrupted the cycle of podocyte injury and mesangial matrix deposition triggered by CXCR4. Podocyte-specific ablation of CXCR4 gene blocked podocyte injury and mesangial cell activation. In vitro, CXCR4 overexpression induced oxidative stress and renin angiotensin system (RAS) activation in podocytes, and triggered the communication between podocytes and mesangial cells. In cultured mesangial cells, AngII treatment induced the expression of SDF-1α, which was secreted into the supernatant to further promote oxidative stress and cell injury in podocytes. Collectively, these results demonstrate that the CXCR4-AT1 axis plays a vital role in glomerular injury via mediating pathologic crosstalk between podocytes and mesangial cells. Our findings uncover a novel pathogenic mechanism by which the CXCR4-AT1 axis promotes glomerular injury.

Long noncoding RNA TUG1 regulates smooth muscle cell differentiation via KLF4-myocardin axis.

Abdominal aortic aneurysms (AAAs) are asymptomatic vascular diseases that have life-threatening outcomes. Smooth muscle cell (SMC) dysfunction plays an important role in AAA development. The contribution of non-coding genome, specifically the role of long non-coding RNAs (lncRNAs) in SMC dysfunction, is relatively unexplored. We investigated the role of lncRNA TUG1 in SMC dysfunction. To identify potential lncRNAs relevant to SMC functionality, lncRNA profiling was performed in angiotensin-II-treated SMCs. AAA was induced by angiotensin-II treatment in mice. Transcriptional regulation of TUG1 was studied using promoter luciferase and chromatin-immuno-precipitation experiments. Gain-or-loss-of-function experiments were performed in vitro to investigate TUG1-mediated regulation of SMC function. Immunoprecipitation experiments were conducted to elucidate the mechanism underlying TUG1-mediated SMC dysfunction. TUG1 was upregulated in SMCs following angiotensin-II treatment. Similarly, TUG1 levels were elevated in abdominal aorta in a mouse model of angiotensin-II-induced AAA. Further investigations showed that angiotensin-II-induced TUG1 expression could be suppressed by inhibiting Notch-signaling pathway, both in vitro and in mouse AAA model and that TUG1 is a direct transcriptional target of the Notch pathway. In aneurysmal tissues, TUG1 expression was inversely correlated with the expression of SMC contractile genes. Overexpression of TUG1 repressed SMC differentiation in vitro, whereas siRNA/shRNA-mediated TUG1 knockdown showed an opposite effect. Mechanistically, TUG1 interacts with transcriptional repressor KLF4 and facilitates its recruitment to myocardin promoter ultimately leading to the repression of SMC differentiation. In summary, our study uncovers a novel role for the lncRNA TUG1 wherein it modulates SMC differentiation via the KLF4-myocardin axis, which may have potential implications in AAA development.NEW & NOTEWORTHY TUG1 is an angiotensin-II-induced long noncoding RNA that mediates smooth muscle cell (SMC) dysfunction through interaction with transcriptional repressor KLF4.

Integrin beta-like 1 mediates fibroblast-cardiomyocyte crosstalk to promote cardiac fibrosis and hypertrophy.

Crosstalk between fibroblasts and cardiomyocytes (CMs) plays a critical role in cardiac remodelling during heart failure (HF); however, the underlying molecular mechanisms remain obscure. Recently, a secretory protein, Integrin beta-like 1 (ITGBL1) was revealed to have detrimental effects on several diseases, such as tumours, pulmonary fibrosis, and hepatic fibrosis; whereas the effect of ITGBL1 on HF is unclear. The purpose of this study was to evaluate its contribution to volume overload-induced remodelling. In this study, we identified ITGBL1 was highly expressed in varied heart diseases and validated in our TAC mice model, especially in fibroblasts. To investigate the role of ITGBL1 in in vitro cell experiments, neonatal rat fibroblasts (NRCFs) and cardiomyocytes (NRCMs) were performed for further study. We found that in comparison to NRCMs, NRCFs expressed high levels of ITGBL1. Meanwhile, ITGBL1 was upregulated in NRCFs, but not in NRCMs following angiotensin-II (AngII) or phenylephrine stimulation. Furthermore, ITGBL1 overexpression promoted NRCFs activation, whereas knockdown of ITGBL1 alleviated NRCFs activation under AngII treatment. Moreover, NRCFs-secreted ITGBL1 could induce NRCMs hypertrophy. Mechanically, ITGBL1-NME/NM23 nucleoside diphosphate kinase 1 (NME1)-TGF-β-Smad2/3 and Wnt signalling pathways were identified to mediate NRCFs activation and NRCMs hypertrophy, respectively. Finally, the knockdown of ITGBL1 in mice subjected to transverse aortic constriction (TAC) surgery recapitulated the in vitro findings, demonstrating blunted cardiac fibrosis, hypertrophy, and improved cardiac function. ITGBL1 is an important functional mediator between fibroblast-cardiomyocyte crosstalk and could be an effective target for cardiac remodelling in HF patients.

Liraglutide Protects Against Diastolic Dysfunction and Improves Ventricular Protein Translation.

Diastolic dysfunction is an increasingly common cardiac pathology linked to heart failure with preserved ejection fraction. Previous studies have implicated glucagon-like peptide 1 (GLP-1) receptor agonists as potential therapies for improving diastolic dysfunction. In this study, we investigate the physiologic and metabolic changes in a mouse model of angiotensin II (AngII)-mediated diastolic dysfunction with and without the GLP-1 receptor agonist liraglutide (Lira). Mice were divided into sham, AngII, or AngII+Lira therapy for 4 weeks. Mice were monitored for cardiac function, weight change, and blood pressure at baseline and after 4 weeks of treatment. After 4 weeks of treatment, tissue was collected for histology, protein analysis, targeted metabolomics, and protein synthesis assays. AngII treatment causes diastolic dysfunction when compared to sham mice. Lira partially prevents this dysfunction. The improvement in function in Lira mice is associated with dramatic changes in amino acid accumulation in the heart. Lira mice also have improved markers of protein translation by Western blot and increased protein synthesis by puromycin assay, suggesting that increased protein turnover protects against fibrotic remodeling and diastolic dysfunction seen in the AngII cohort. Lira mice also lost lean muscle mass compared to the AngII cohort, raising concerns about peripheral muscle scavenging as a source of the increased amino acids in the heart. Lira therapy protects against AngII-mediated diastolic dysfunction, at least in part by promoting amino acid uptake and protein turnover in the heart. Liraglutide therapy is associated with loss of mean muscle mass, and long-term studies are warranted to investigate sarcopenia and frailty with liraglutide therapy in the setting of diastolic disease.

Sesamin suppresses angiotensin-II-enhanced oxidative stress and hypertrophic markers in H9c2 cells.

Myocardial hypertrophy plays a crucial role in cardiovascular disease (CVD) development. Myocardial hypertrophy is an adaptive response by myocardial cells to stress after cardiac injury to maintain cardiac output and function. Angiotensin II (Ang-II) regulates CVD through the renin-angiotensin-aldosterone system, and its signaling in cardiac myocytes leads to excessive reactive oxygen species (ROS) production, oxidative stress, and inflammation. Sesamin (SA), a natural compound in sesame seeds, has anti-inflammatory and anti-apoptotic effects. This study investigated whether SA could attenuate hypertrophic damage and oxidative injuries in H9c2 cells under Ang-II stimulation. We found that SA decreased the cell surface area. Furthermore, Ang-II treatment reduced Ang-II-increased ANP, BNP, and β-MHC expression. Ang-II enhanced NADPH oxidase activity, ROS formation, and decreased Superoxide Dismutase (SOD) activity. SA treatment reduces Ang-II-caused oxidative injuries. We also found that SA mitigates Ang-II-induced apoptosis and pro-inflammatory responses. In conclusion, SA could attenuate Ang-II-induced cardiac hypertrophic injuries by inhibiting oxidative stress, apoptosis, and inflammation in H9c2 cells. Therefore, SA might be a potential supplement for CVD management.