Counteracting Angiotensinogen Small-Interfering RNA-Mediated Antihypertensive Effects With REVERSIR.

BACKGROUND: Ssmall-interfering RNA (siRNA) targeting hepatic AGT (angiotensinogen) mRNA depletes AGT, lowering blood pressure for up to 6 months. However, certain situations may require a rapid angiotensin increase. The reverse siRNA silencing, RVR technology is a potential approach to counteract siRNA effects. METHODS: Spontaneously hypertensive rats received 10 mg/kg AGT siRNA, and 3 weeks later were given AGT-RVR (1, 10, or 20 mg/kg). One week after AGT-RVR dosing, a redose of AGT siRNA assessed its post-AGT-RVR effectiveness for 2 weeks. Additionally, the impact of AGT-RVR after an equihypotensive dose of valsartan (4 mg/kg per day) was examined. RESULTS: Baseline mean arterial pressure (MAP) was 144±1 mm Hg. AGT siRNA reduced MAP by ≈16 mm Hg and AGT by >95%, while renin increased 25-fold. All AGT-RVR doses restored MAP to baseline within 4 to 7 days. Notably, 10 and 20 mg/kg restored AGT and renin to baseline, while 1 mg/kg allowed ≈50% AGT restoration, with renin remaining above baseline. A second AGT siRNA treatment, following 1-mg/kg AGT-RVR, reduced MAP to the same degree as the initial dose, while following 10 mg/kg AGT-RVR, it resulted in ≈50% of the first dose's MAP effect at 2 weeks. The valsartan-induced MAP reduction was unaffected by AGT-RVR. CONCLUSIONS: In spontaneously hypertensive rats, angiotensinogen-RVR dose-dependently reversed AGT siRNA-induced AGT reduction, normalizing MAP. MAP normalization persisted even with 50% recovered AGT levels, likely due to upregulated renin maintaining adequate angiotensin generation. Post-AGT-RVR dosing, a second AGT siRNA dose lowered MAP again.

Intracellular pathways of calcitonin gene-related peptide-induced relaxation of human coronary arteries: A key role for Gßγ subunit instead of cAMP.

BACKGROUND AND PURPOSE: Calcitonin gene-related peptide (CGRP) is a potent vasodilator. While its signalling is assumed to be mediated via increases in cAMP, this study focused on elucidating the actual intracellular signalling pathways involved in CGRP-induced relaxation of human isolated coronary arteries (HCA). EXPERIMENTAL APPROACH: HCA were obtained from heart valve donors (27 M, 25 F, age 54 ± 2 years). Concentration-response curves to human α-CGRP or forskolin were constructed in HCA segments, incubated with different inhibitors of intracellular signalling pathways, and intracellular cAMP levels were measured with and without stimulation. RESULTS: Adenylyl cyclase (AC) inhibitors SQ22536 + DDA and MDL-12330A, and PKA inhibitors Rp-8-Br-cAMPs and H89, did not inhibit CGRP-induced relaxation of HCA, nor did the guanylyl cyclase inhibitor ODQ, PKG inhibitor KT5823, EPAC1/2 inhibitor ESI09, potassium channel blockers TRAM-34 + apamin, iberiotoxin or glibenclamide, or the Gαq inhibitor YM-254890. Phosphodiesterase inhibitors induced a concentration-dependent decrease in the response to KCl but did not potentiate relaxation to CGRP. Relaxation to forskolin was not blocked by PKA or AC inhibitors, although AC inhibitors significantly inhibited the increase in cAMP. Inhibition of Gßγ subunits using gallein significantly inhibited the relaxation to CGRP in human coronary arteries. CONCLUSION: While CGRP signalling is generally assumed to act via cAMP, the CGRP-induced vasodilation in HCA was not inhibited by targeting this intracellular signalling pathway at different levels. Instead, inhibition of Gßγ subunits did inhibit the relaxation to CGRP, suggesting a different mechanism of CGRP-induced relaxation than generally believed.

Functional Analysis of TRPA1, TRPM3, and TRPV1 Channels in Human Dermal Arteries and Their Role in Vascular Modulation.

Transient receptor potential (TRP) channels are pivotal in modulating vascular functions. In fact, topical application of cinnamaldehyde or capsaicin (TRPA1 and TRPV1 channel agonists, respectively) induces "local" changes in blood flow by releasing vasodilator neuropeptides. We investigated TRP channels' contributions and the pharmacological mechanisms driving vasodilation in human isolated dermal arteries. Ex vivo studies assessed the vascular function of artery segments and analyzed the effects of different compounds. Concentration-response curves to cinnamaldehyde, pregnenolone sulfate (PregS, TRPM3 agonist), and capsaicin were constructed to evaluate the effect of the antagonists HC030031 (TRPA1); isosakuranetin (TRPM3); and capsazepine (TRPV1). Additionally, the antagonists/inhibitors olcegepant (CGRP receptor); L-NAME (nitric oxide synthase); indomethacin (cyclooxygenase); TRAM-34 plus apamin (K+ channels); and MK-801 (NMDA receptors, only for PregS) were used. Moreover, CGRP release was assessed in the organ bath fluid post-agonist-exposure. In dermal arteries, cinnamaldehyde- and capsaicin-induced relaxation remained unchanged after the aforementioned antagonists, while PregS-induced relaxation was significantly inhibited by isosakuranetin, L-NAME and MK-801. Furthermore, there was a significant increase in CGRP levels post-agonist-exposure. In our experimental model, TRPA1 and TRPV1 channels seem not to be involved in cinnamaldehyde- or capsaicin-induced relaxation, respectively, whereas TRPM3 channels contribute to PregS-induced relaxation, possibly via CGRP-independent mechanisms.

Combining renin-angiotensin system blockade and sodium-glucose cotransporter-2 inhibition in experimental diabetes results in synergistic beneficial effects.

BACKGROUND: Sodium-glucose cotransporter-2 (SGLT2) inhibition exerts cardioprotective and renoprotective effects, often on top of renin-angiotensin system (RAS) blockade. We investigated this in diabetic hypertensive (mREN2)27 rats. METHODS: Rats were made diabetic with streptozotocin and treated with vehicle, the angiotensin receptor blocker valsartan, the SGLT2 inhibitor empagliflozin, or their combination. Blood pressure (BP) was measured by telemetry. RESULTS: Diabetes resulted in albuminuria, accompanied by glomerulosclerosis, without a change in glomerular filtration rate. Empagliflozin did not lower BP, while valsartan did, and when combined the BP drop was largest. Only dual blockade reduced cardiac hypertrophy and prevented left ventricular dilatation. Valsartan, but not empagliflozin, increased renin, and the largest renin rise occurred during dual blockade, resulting in plasma angiotensin II [but not angiotensin-(1-7)] upregulation. In contrast, in the kidney, valsartan lowered angiotensin II and angiotensin-(1-7), and empagliflozin did not alter this. Although both valsartan and empagliflozin alone tended to diminish albuminuria, the reduction was significant only when both drugs were combined. This was accompanied by reduced glomerulosclerosis, no change in glomerular filtration rate, and a favorable expression pattern of fibrosis and inflammatory markers (including SGLT2) in the kidney. CONCLUSION: RAS blockade and SGLT2 inhibition display synergistic beneficial effects on BP, kidney injury and cardiac hypertrophy in a rat with hypertension and diabetes. The synergy does not involve upregulation of angiotensin-(1-7), but may relate to direct RAS-independent effects of empagliflozin in the heart and kidney.

Cyclooxygenase-2 inhibition prevents renal toxicity but not hypertension during sunitinib treatment.

BACKGROUND: Anticancer angiogenesis inhibitors cause hypertension and renal injury. Previously we observed in rats that high-dose aspirin (capable of blocking cyclooxygenase (COX)-1 and-2) was superior to low-dose aspirin (blocking COX-1 only) to prevent these side-effects during treatment with the angiogenesis inhibitor sunitinib, suggesting a role for COX-2. High-dose aspirin additionally prevented the rise in COX-derived prostacyclin (PGI2). Therefore, we studied the preventive effects of selective COX-2 inhibition and the hypothesized contributing role of PGI2 during angiogenesis inhibition. METHODS: Male WKY rats received vehicle, sunitinib ((SU), 14 mg/kg/day) alone or combined with COX-2 inhibition (celecoxib, 10 mg/kg/day) or a PGI2 analogue (iloprost, 100 µg/kg/day) for 8 days (n = 8-9 per group). Mean arterial pressure (MAP) was measured via radiotelemetry, biochemical measurements were performed via ELISA and vascular function was assessed via wire myography. RESULTS: SU increased MAP (17±1mmHg versus 3±1mmHg after vehicle on day 4, P < 0.002), which could not be significantly blunted by celecoxib (+12±3mmHg on day 4, P = 0.247), but was temporarily attenuated by iloprost (treatment days 1 + 2 only). Urinary PGI2 (996 ± 112 versus 51 ± 11ng/24h after vehicle, P < 0.001), but not circulating PGI2 increased during SU, which remained unaffected by celecoxib and iloprost. Celecoxib reduced sunitinib-induced albuminuria (0.36 ± 0.05 versus 0.58 ± 0.05mg/24h after SU, P = 0.005). Wire myography demonstrated increased vasoconstriction to endothelin-1 after SU (Emax P = 0.005 versus vehicle), which remained unaffected by celecoxib or iloprost. CONCLUSION: Selective COX-2 inhibition ameliorates albuminuria during angiogenesis inhibition with sunitinib, which most likely acts independently of PGI2. To combat angiogenesis inhibitor-induced hypertension, dual rather than selective COX-1/2 blockade seems preferential.

Dietary sodium restriction prevents vascular endothelial growth factor inhibitor-induced hypertension.

BACKGROUND: Vascular endothelial growth factor inhibitors (VEGFIs) are effective anticancer agents which often induce hypertension. VEGFI-induced hypertension is sodium-sensitive in animal studies. Therefore, the efficacy of dietary sodium restriction (DSR) to prevent VEGFI-induced hypertension in cancer patients was studied. METHODS: Cancer patients with VEGFI-induced hypertension (day mean >135/85 mmHg or a rise in systolic and/or diastolic BP ≥ 20 mmHg) were treated with DSR (aiming at <4 g salt/day). The primary endpoint was the difference in daytime mean arterial blood pressure (MAP) increase between the treatment cycle with and without DSR. RESULTS: During the first VEGFI treatment cycle without DSR, mean daytime MAP increased from 95 to 110 mmHg. During the subsequent treatment cycle with DSR, mean daytime MAP increased from 94 to 102 mmHg. Therefore, DSR attenuated the increase in mean daytime MAP by 7 mmHg (95% CI 1.3-12.0, P = 0.009). DSR prevented the rise in the endothelin-1/renin ratio that normally accompanies VEGFI-induced hypertension (P = 0.020) and prevented the onset of proteinuria: 0.15 (0.10-0.25) g/24 h with DSR versus 0.19 (0.11-0.32) g/24 h without DSR; P = 0.005. DISCUSSION: DSR significantly attenuated VEGFI induced BP rise and proteinuria and thus is an effective non-pharmacological intervention.

Blood pressure-independent renoprotective effects of small interference RNA targeting liver angiotensinogen in experimental diabetes.

BACKGROUND AND PURPOSE: Small interfering RNA (siRNA) targeting liver angiotensinogen lowers blood pressure, but its effects in hypertensive diabetes are unknown. EXPERIMENTAL APPROACH: To address this, TGR (mRen2)27 rats (angiotensin II-dependent hypertension model) were made diabetic with streptozotocin over 18 weeks and treated with either vehicle, angiotensinogen siRNA, the AT1 antagonist valsartan, the ACE inhibitor captopril, valsartan + siRNA or valsartan + captopril for the final 3 weeks. Mean arterial pressure (MAP) was measured via radiotelemetry. KEY RESULTS: MAP before treatment was 153 ± 2 mmHg. Diabetes resulted in albuminuria, accompanied by glomerulosclerosis and podocyte effacement, without a change in glomerular filtration rate. All treatments lowered MAP and cardiac hypertrophy, and the largest drop in MAP was observed with siRNA + valsartan. Treatment with siRNA lowered circulating angiotensinogen by >99%, and the lowest circulating angiotensin II and aldosterone levels occurred in the dual treatment groups. Angiotensinogen siRNA did not affect renal angiotensinogen mRNA expression, confirming its liver-specificity. Furthermore, only siRNA with or without valsartan lowered renal angiotensin I. All treatments lowered renal angiotensin II and the reduction was largest (>95%) in the siRNA + valsartan group. All treatments identically lowered albuminuria, whereas only siRNA with or without valsartan restored podocyte foot processes and reduced glomerulosclerosis. CONCLUSION AND IMPLICATIONS: Angiotensinogen siRNA exerts renoprotection in diabetic TGR (mRen2)27 rats and this relies, at least in part, on the suppression of renal angiotensin II formation from liver-derived angiotensinogen. Clinical trials should now address whether this is also beneficial in human diabetic kidney disease.

An explorative epigenome-wide association study of plasma renin and aldosterone concentration in a Ghanaian population: the RODAM study.

BACKGROUND: The epigenetic regulation of the renin-angiotensin-aldosterone system (RAAS) potentially plays a role in the pathophysiology underlying the high burden of hypertension in sub-Saharan Africans (SSA). Here we report the first epigenome-wide association study (EWAS) of plasma renin and aldosterone concentrations and the aldosterone-to-renin ratio (ARR). METHODS: Epigenome-wide DNA methylation was measured using the Illumina 450K array on whole blood samples of 68 Ghanaians. Differentially methylated positions (DMPs) were assessed for plasma renin concentration, aldosterone, and ARR using linear regression models adjusted for age, sex, body mass index, diabetes mellitus, hypertension, and technical covariates. Additionally, we extracted methylation loci previously associated with hypertension, kidney function, or that were annotated to RAAS-related genes and associated these with renin and aldosterone concentration. RESULTS: We identified one DMP for renin, ten DMPs for aldosterone, and one DMP associated with ARR. Top DMPs were annotated to the PTPRN2, SKIL, and KCNT1 genes, which have been reported in relation to cardiometabolic risk factors, atherosclerosis, and sodium-potassium handling. Moreover, EWAS loci previously associated with hypertension, kidney function, or RAAS-related genes were also associated with renin, aldosterone, and ARR. CONCLUSION: In this first EWAS on RAAS hormones, we identified DMPs associated with renin, aldosterone, and ARR in a SSA population. These findings are a first step in understanding the role of DNA methylation in regulation of the RAAS in general and in a SSA population specifically. Replication and translational studies are needed to establish the role of these DMPs in the hypertension burden in SSA populations.

Serum CGRP in migraine patients using erenumab as preventive treatment.

AIM: Serum levels of Calcitonin Gene-Related Peptide (CGRP)-like immunoreactivity (CGRP-LI) in migraine patients before and after starting treatment with erenumab were measured to evaluate the association with clinical treatment response. METHODS: Blood samples were collected from the cubital fossa before (T0) and 2-4 weeks after (T1) starting treatment with erenumab. Clinical response was monitored using a daily headache e-diary. Serum levels of CGRP-LI, assessed using radioimmunoassay, were compared between T0 and T1, correcting for migraine reduction. In addition, for both T0 and T1, linear regression models were constructed using migraine reduction as outcome and serum CGRP-LI as independent variable, corrected for age, gender and monthly migraine days (MMD) at baseline. RESULTS: Serum CGRP-LI did not differ between T0 and T1 (p = 0.30). However, there was an interaction between time and reduction in MMD (p = 0.01). Absolute reduction in MMD in the third month after treatment with erenumab was associated with serum CGRP-LI at T1, 2-4 weeks after starting treatment with erenumab (p = 0.003), but not with serum CGRP-LI at T0 (p = 0.24). CONCLUSION: Lower serum CGRP-LI 2-4 weeks after starting treatment with erenumab was associated with a higher reduction in migraine days after three months of treatment. Although the underlying mechanisms remain to be determined, this suggests that changes in CGRP levels, shortly after starting erenumab, are important for its clinical effect.

Conventional Vasopressor and Vasopressor-Sparing Strategies to Counteract the Blood Pressure-Lowering Effect of Small Interfering RNA Targeting Angiotensinogen.

Background A single dose of small interfering RNA (siRNA) targeting liver angiotensinogen eliminates hepatic angiotensinogen and lowers blood pressure. Angiotensinogen elimination raises concerns for clinical application because an angiotensin rise is needed to maintain perfusion pressure during hypovolemia. Here, we investigated whether conventional vasopressors can raise arterial pressure after angiotensinogen depletion. Methods and Results Spontaneously hypertensive rats on a low-salt diet were treated with siRNA (10 mg/kg fortnightly) for 4 weeks, supplemented during the final 2 weeks with fludrocortisone (6 mg/kg per day), the α-adrenergic agonist midodrine (4 mg/kg per day), or a high-salt diet (all groups n=6-7). Pressor responsiveness to angiotensin II and norepinephrine was assessed before and after siRNA administration. Blood pressure was measured via radiotelemetry. Depletion of liver angiotensinogen by siRNA lowered plasma angiotensinogen concentrations by 99.2±0.1% and mean arterial pressure by 19 mm Hg. siRNA-mediated blood pressure lowering was rapidly reversed by intravenous angiotensin II or norepinephrine, or gradually reversed by fludrocortisone or high salt intake. Midodrine had no effect. Unexpectedly, fludrocortisone partially restored plasma angiotensinogen concentrations in siRNA-treated rats, and nearly abolished plasma renin concentrations. To investigate whether this angiotensinogen originated from nonhepatic sources, fludrocortisone was administered to mice lacking hepatic angiotensinogen. Fludrocortisone did not increase angiotensinogen in these mice, implying that the rise in angiotensinogen in the siRNA-treated rats must have depended on the liver, most likely reflecting diminished cleavage by renin. Conclusions Intact pressor responsiveness to conventional vasopressors provides pharmacological means to regulate the blood pressure-lowering effect of angiotensinogen siRNA and may support future therapeutic implementation of siRNA.

Differential effects of cyclo-oxygenase 1 and 2 inhibition on angiogenesis inhibitor-induced hypertension and kidney damage.

Vascular endothelial growth factor antagonism with angiogenesis inhibitors in cancer patients induces a 'preeclampsia-like' syndrome including hypertension, proteinuria and elevated endothelin (ET)-1. Cyclo-oxygenase (COX) inhibition with aspirin is known to prevent the onset of preeclampsia in high-risk patients. In the present study, we hypothesised that treatment with aspirin would prevent the development of angiogenesis inhibitor-induced hypertension and kidney damage. Our aims were to compare the effects of low-dose (COX-1 inhibition) and high-dose (dual COX-1 and COX-2 inhibition) aspirin on blood pressure, vascular function, oxidative stress, ET-1 and prostanoid levels and kidney damage during angiogenesis-inhibitor therapy in rodents. To this end, Wistar Kyoto rats were treated with vehicle, angiogenesis inhibitor (sunitinib) alone or in combination with low- or high-dose aspirin for 8 days (n=5-7/group). Our results demonstrated that prostacyclin (PGI2) and ET-1 were increased during angiogenesis-inhibitor therapy, while thromboxane (TXA2) was unchanged. Both low- and high-dose aspirin blunted angiogenesis inhibitor-induced hypertension and vascular superoxide production to a similar extent, whereas only high-dose aspirin prevented albuminuria. While circulating TXA2 and prostaglandin F2α levels were reduced by both low- and high-dose aspirin, circulating and urinary levels PGI2 were only reduced by high-dose aspirin. Lastly, treatment with aspirin did not significantly affect ET-1 or vascular function. Collectively our findings suggest that prostanoids contribute to the development of angiogenesis inhibitor-induced hypertension and renal damage and that targeting the prostanoid pathway could be an effective strategy to mitigate the unwanted cardiovascular and renal toxicities associated with angiogenesis inhibitors.

The relation of RAAS activity and endothelin-1 levels to coronary atherosclerotic burden and microvascular dysfunction in chest pain patients.

BACKGROUND AND AIMS: In this study, we investigated whether increased renin angiotensin aldosterone system (RAAS) activation and endothelin-1 levels are related to coronary artery calcium (CAC) score, total plaque volume (TPV), high risk plaque, hyperemic myocardial blood flow (MBF) and coronary microvascular dysfunction (CMD). METHODS: In a prospective, observational, cross-sectional cohort, renin as a marker for RAAS activation and endothelin-1 were measured in peripheral venous blood of 205 patients (64% men; age 58 ± 8.7 years) with suspected coronary artery disease (CAD) who underwent coronary computed tomography angiography (CCTA), [15O]H2O positron emission tomography (PET) perfusion imaging and invasive fractional flow reserve (FFR) measurements. Patients were categorized into three groups based on FFR (≤0.80) and hyperemic MBF <2.3 ml/min/g: [1] obstructive CAD (n = 92), [2] CMD (n = 26) or [3] no or non-obstructive CAD (n = 85). RESULTS: After correction for baseline characteristics, including RAAS inhibiting therapy, renin associated positively with CAC score and TPV, but not with hyperemic MBF (p < 0.01; p = 0.02 and p = 0.23). Patients with high risk plaque displayed higher levels of renin (mean logarithmic renin 1.25 ± 0.43 vs. 1.12 ± 0.35 pg/ml; p = 0.04), but not endothelin-1. Compared to no or non-obstructive CAD patients, renin was significantly elevated in obstructive CAD patients but not in CMD patients (mean logarithmic renin 1.06 ± 0.34 vs. 1.23 ± 0.36; p < 0.01 and 1.06 ± 0.34 vs. 1.16 ± 0.41 pg/ml; p = 0.65). Endothelin-1 did not differ between the three patient groups. CONCLUSIONS: Our report provides evidence that RAAS activity measured by renin concentration is elevated in patients with coronary atherosclerosis and high risk plaque but not in patients with CMD, whereas endothelin-1 is not related to either.

A low aldosterone/renin ratio and high soluble ACE2 associate with COVID-19 severity.

BACKGROUND: The severity of COVID-19 after SARS-CoV-2 infection is unpredictable. Angiotensin-converting enzyme-2 (ACE2) is the receptor responsible for coronavirus binding, while subsequent cell entry relies on priming by the serine protease TMPRSS2 (transmembrane protease, serine 2). Although renin-angiotensin-aldosterone-system (RAAS) blockers have been suggested to upregulate ACE2, their use in COVID-19 patients is now considered well tolerated. The aim of our study was to investigate parameters that determine COVID-19 severity, focusing on RAAS-components and variation in the genes encoding for ACE2 and TMPRSS2. METHODS: Adult patients hospitalized due to SARS-CoV-2 infection between May 2020 and October 2020 in the Haga Teaching Hospital were included, and soluble ACE2 (sACE2), renin, aldosterone (in heparin plasma) and polymorphisms in the ACE2 and TMPRSS2 genes (in DNA obtained from EDTA blood) were determined. MEASUREMENTS AND MAIN RESULTS: Out of the 188 patients who were included, 60 were defined as severe COVID-19 (ICU and/or death). These patients more often used antidiabetic drugs, were older, had higher renin and sACE2 levels, lower aldosterone levels and a lower aldosterone/renin ratio. In addition, they displayed the TMPRSS2-rs2070788 AA genotype less frequently. No ACE2 polymorphism-related differences were observed. Multivariate regression analysis revealed independent significance for age, sACE2, the aldosterone/renin ratio, and the TMPRSS2 rs2070788 non-AA genotype as predictors of COVID-19 severity, together yielding a C-index of 0.79. Findings were independent of the use of RAAS blockers. CONCLUSION: High sACE2, a low aldosterone/renin ratio and having the TMPRSS2 rs2070788 non-AA genotype are novel independent determinants that may help to predict COVID-19 disease severity. TRIAL REGISTRATION: retrospectively registered.

In Vivo Renin Activity Imaging in the Kidney of Progeroid Ercc1 Mutant Mice.

Changes in the renin-angiotensin system, known for its critical role in the regulation of blood pressure and sodium homeostasis, may contribute to aging and age-related diseases. While the renin-angiotensin system is suppressed during aging, little is known about its regulation and activity within tissues. However, this knowledge is required to successively treat or prevent renal disease in the elderly. Ercc1 is involved in important DNA repair pathways, and when mutated causes accelerated aging phenotypes in humans and mice. In this study, we hypothesized that unrepaired DNA damage contributes to accelerated kidney failure. We tested the use of the renin-activatable near-infrared fluorescent probe ReninSense680™ in progeroid Ercc1d/- mice and compared renin activity levels in vivo to wild-type mice. First, we validated the specificity of the probe by detecting increased intrarenal activity after losartan treatment and the virtual absence of fluorescence in renin knock-out mice. Second, age-related kidney pathology, tubular anisokaryosis, glomerulosclerosis and increased apoptosis were confirmed in the kidneys of 24-week-old Ercc1d/- mice, while initial renal development was normal. Next, we examined the in vivo renin activity in these Ercc1d/- mice. Interestingly, increased intrarenal renin activity was detected by ReninSense in Ercc1d/- compared to WT mice, while their plasma renin concentrations were lower. Hence, this study demonstrates that intrarenal RAS activity does not necessarily run in parallel with circulating renin in the aging mouse. In addition, our study supports the use of this probe for longitudinal imaging of altered RAS signaling in aging.

Selective Phosphodiesterase 1 Inhibition Ameliorates Vascular Function, Reduces Inflammatory Response, and Lowers Blood Pressure in Aging Animals.

Diminished nitric oxide-cGMP-mediated relaxation plays a crucial role in cardiovascular aging, leading to decreased vasodilation, vascular hypertrophy and stiffening, and ultimately, cardiovascular dysfunction. Aging is the time-related worsening of physiologic function due to complex cellular and molecular interactions, and it is at least partly driven by DNA damage. Genetic deletion of the DNA repair enzyme ERCC1 endonuclease in Ercc1Δ/- mice provides us an efficient tool to accelerate vascular aging, explore mechanisms, and test potential treatments. Previously, we identified the cGMP-degrading enzyme phosphodiesterase 1 as a potential treatment target in vascular aging. In the present study, we studied the effect of acute and chronic treatment with ITI-214, a selective phosphodiesterase 1 inhibitor on vascular aging features in Ercc1Δ/- mice. Compared with wild-type mice, Ercc1Δ/- mice at the age of 14 weeks showed decreased reactive hyperemia, diminished endothelium-dependent and -independent responses of arteries in organ baths, carotid wall hypertrophy, and elevated circulating levels of inflammatory cytokines. Acute ITI-214 treatment in organ baths restored the arterial endothelium-independent vasodilation in Ercc1Δ/- mice. An 8-week treatment with 100 mg/kg per day ITI-214 improved endothelium-independent relaxation in both aorta and coronary arteries, at least partly restored the diminished reactive hyperemia, lowered the systolic and diastolic blood pressure, normalized the carotid hypertrophy, and ameliorated inflammatory responses exclusively in Ercc1Δ/- mice. These findings suggest phosphodiesterase 1 inhibition would provide a powerful tool for nitric oxide-cGMP augmentation and have significant therapeutic potential to battle arteriopathy related to aging. SIGNIFICANCE STATEMENT: The findings implicate the key role of phosphodiesterase 1 in vascular function and might be of clinical importance for the prevention of mortalities and morbidities related to vascular complications during aging, as well as for patients with progeria that show a high risk of cardiovascular disease.

Renoprotective Effects of Small Interfering RNA Targeting Liver Angiotensinogen in Experimental Chronic Kidney Disease.

[Figure: see text].

No evidence for brain renin-angiotensin system activation during DOCA-salt hypertension.

Brain renin-angiotensin system (RAS) activation is thought to mediate deoxycorticosterone acetate (DOCA)-salt hypertension, an animal model for human primary hyperaldosteronism. Here, we determined whether brainstem angiotensin II is generated from locally synthesized angiotensinogen and mediates DOCA-salt hypertension. To this end, chronic DOCA-salt-hypertensive rats were treated with liver-directed siRNA targeted to angiotensinogen, the angiotensin II type 1 receptor antagonist valsartan, or the mineralocorticoid receptor antagonist spironolactone (n = 6-8/group). We quantified circulating angiotensinogen and renin by enzyme-kinetic assay, tissue angiotensinogen by Western blotting, and angiotensin metabolites by LC-MS/MS. In rats without DOCA-salt, circulating angiotensin II was detected in all rats, whereas brainstem angiotensin II was detected in 5 out of 7 rats. DOCA-salt increased mean arterial pressure by 19 ± 1 mmHg and suppressed circulating renin and angiotensin II by >90%, while brainstem angiotensin II became undetectable in 5 out of 7 rats (<6 fmol/g). Gene silencing of liver angiotensinogen using siRNA lowered circulating angiotensinogen by 97 ± 0.3%, and made brainstem angiotensin II undetectable in all rats (P<0.05 vs. non-DOCA-salt), although brainstem angiotensinogen remained intact. As expected for this model, neither siRNA nor valsartan attenuated the hypertensive response to DOCA-salt, whereas spironolactone normalized blood pressure and restored brain angiotensin II together with circulating renin and angiotensin II. In conclusion, despite local synthesis of angiotensinogen in the brain, brain angiotensin II depended on circulating angiotensinogen. That DOCA-salt suppressed circulating and brain angiotensin II in parallel, while spironolactone simultaneously increased brain angiotensin II and lowered blood pressure, indicates that DOCA-salt hypertension is not mediated by brain RAS activation.

The Effects of Acute and Chronic Selective Phosphodiesterase 1 Inhibition on Smooth Muscle Cell-Associated Aging Features.

Age-related cardiovascular diseases (CVDs) remain among the leading global causes of death, and vascular smooth muscle cell (VSMC) remodeling plays an essential role in its pathology. Reduced NO-cGMP pathway signaling is a major feature and pathogenic mechanism underlying vasodilator dysfunction. Recently, we identified phosphodiesterase (PDE) 1, an enzyme that hydrolyzes and inactivates the cyclic nucleotides cAMP and cGMP, and thereby provides a potential treatment target for restoring age-related vascular dysfunction due to aging of VSMC. Based on this hypothesis, we here tested the effects of PDE1 inhibition in a model of SMC-specific accelerated aging mice. SMC-KO and their WT littermates received either vehicle or the PDE1 inhibitor lenrispodun for 8 weeks. Vascular function was measured both in vivo (Laser Doppler technique) and ex vivo (organ bath). Moreover, we deployed UV irradiation in cell culture experiments to model accelerated aging in an in vitro situation. SMC-KO mice display a pronounced loss of vasodilator function in the isolated aorta, the cutaneous microvasculature, and mesenteric arteries. Ex vivo, in isolated vascular tissue, we found that PDE1 inhibition with lenrispodun improves vasodilation, while no improvement was observed in isolated aorta taken from mice after chronic treatment in vivo. However, during lenrispodun treatment in vivo, an enhanced microvascular response in association with upregulated cGMP levels was seen. Further, chronic lenrispodun treatment decreased TNF-α and IL-10 plasma levels while the elevated level of IL-6 in SMC-KO mice remained unchanged after treatment. PDE1 and senescence markers, p16 and p21, were increased in both SMC-KO aorta and cultured human VSMC in which DNA was damaged by ultraviolet irradiation. This increase was lowered by chronic lenrispodun. In contrast, lenrispodun increased the level of PDE1A in both situations. In conclusion, we demonstrated that PDE1 inhibition may be therapeutically useful in reversing aspects of age-related VSMC dysfunction by potentiating NO-cGMP signaling, preserving microvascular function, and decreasing senescence. Yet, after chronic treatment, the effects of PDE1 inhibition might be counteracted by the interplay between differential PDE1A and C expression. These results warrant further pharmacodynamic profiling of PDE enzyme regulation during chronic PDE1 inhibitor treatment.