Amlodipine limits microglia activation and cognitive dysfunction in aged hypertensive mice.

BACKGROUND: SBP and blood pressure variability are independent risk factors for cerebral small vessel disease, a leading cause for stroke and dementia. Calcium-channel blockers are known to reduce blood pressure variability and may thus offer benefit against dementia. Beyond this effect, the impact of calcium-channel blockers on hypertension-induced neuroinflammation, and especially, microglial phenotype remains unknown. We aimed to study the ability of amlopidine to alleviate microglia inflammation, and slow down cognitive dysfunction in aged hypertensive mice. METHODS: Hypertensive BPH/2J and normotensive BPN/3J mice were studied until 12 months of age. Hypertensive mice were untreated or received amlodipine (10 mg/kg per day). Blood pressure parameters were measured by telemetry and tail cuff plethysmography. Mice underwent repeated series of cognitive tasks. Brain immunohistochemistry was performed to study blood-brain barrier dysfunction and microglial pro-inflammatory phenotype (CD68 + Iba1 + cells; morphological analysis). RESULTS: Amlodipine normalized SBP over the entire life span and decreased blood pressure variability. BPH/2J mice exhibited impaired short-term memory that was prevented by amlodipine at 12 months (discrimination index 0.41 ±â€Š0.25 in amlodipine-treated vs. 0.14 ±â€Š0.15 in untreated BPH/2J mice, P  = 0.02). Amlopidine treatment of BPH/2J did not prevent blood-brain barrier leakage, a measure of cerebral small vessel disease, but limited its size. Microglia's inflammatory phenotype in BPH/2J, characterized by an increased number of Iba1 + CD68 + cells, increased soma size and shortened processes, was partly reduced by amlodipine. CONCLUSION: Amlodipine attenuated the short-term memory impairment in aged hypertensive mice. Beyond its blood pressure lowering capacity, amlodipine may be cerebroprotective by modulating neuroinflammation.

Pharmacological depletion of microglia and perivascular macrophages prevents Vascular Cognitive Impairment in Ang II-induced hypertension.

Rationale: Hypertension is a major risk factor for cerebral small vessel disease, the most prevalent cause of vascular cognitive impairment. As we have shown, hypertension induced by a prolonged Angiotensin II infusion is associated with increased permeability of the blood-brain barrier (BBB), chronic activation of microglia and myelin loss. In this study we therefore aim to determine the contribution of microglia to hypertension-induced cognitive impairment in an experimental hypertension model by a pharmacological depletion approach. Methods: For this study, adult Cx3Cr1 gfp/wtxThy1 yfp/0 reporter mice were infused for 12 weeks with Angiotensin II or saline and subgroups were treated with PLX5622, a highly selective CSF1R tyrosine kinase inhibitor. Systolic blood pressure (SBP) was measured via tail-cuff. Short- and long-term spatial memory was assessed during an Object Location task and a Morris Water Maze task (MWM). Microglia depletion efficacy was assessed by flow cytometry and immunohistochemistry. BBB leakages, microglia phenotype and myelin integrity were assessed by immunohistochemistry. Results: SBP, heart weight and carotid pulsatility were increased by Ang II and were not affected by PLX5622. Short-term memory was significantly impaired in Ang II hypertensive mice, and partly prevented in Ang II mice treated with PLX5622. Histological and flow cytometry analysis revealed almost complete ablation of microglia and a 60% depletion of brain resident perivascular macrophages upon CSF1R inhibition. Number and size of BBB leakages were increased in Ang II hypertensive mice, but not altered by PLX5622 treatment. Microglia acquired a pro-inflammatory phenotype at the site of BBB leakages in both Saline and Ang II mice and were successfully depleted by PLX5622. There was however no significant change in myelin integrity at the site of leakages. Conclusion: Our results show that depletion of microglia and PVMs, by CSF1R inhibition prevents short-term memory impairment in Ang II induced hypertensive mice. We suggest this beneficial effect is mediated by the major decrease of pro-inflammatory microglia within BBB leakages. This novel finding supports the critical role of brain immune cells in the pathogenesis of hypertension-related cognitive impairment. An adequate modulation of microglia /PVM density and phenotype may constitute a relevant approach to prevent and/or limit the progression of vascular cognitive impairment.

Haemodynamic effects of the flavonoid quercetin in rats revisited.

BACKGROUND AND PURPOSE: The flavonoid quercetin increased the in vitro potency of the α1 -antagonist tamsulosin to reduce phenylephrine-dependent arterial contractions by 10-fold. To examine if this supplement-drug interaction luxates hypotensive and orthostatic events in vivo, several set of studies were conducted in spontaneously hypertensive (SHR) and normotensive (Wistar Kyoto [WKY]) rats. EXPERIMENTAL APPROACH: First, in rats pretreated with quercetin or its vehicle, responses to phenylephrine and tamsulosin were examined. Second, tamsulosin-induced changes in renal, mesenteric, hindquarter and carotid conductance were compared in quercetin- and vehicle-treated rats instrumented with Doppler flow probes. Animals were also placed on a tilt table to record regional haemodynamic changes to orthostatic challenges. Third, adult SHR were instrumented with telemeters to measure 24-hr patterns of BP. Recordings were made before and during a 5-week oral treatment of quercetin. Finally, pre-hypertensive SHR were treated with quercetin from 4 to 8 weeks of age and arterial pressure was measured at 8 and 12 weeks. KEY RESULTS: Pretreatment with quercetin did not influence the responses to phenylephrine and tamsulosin, in neither WKY nor SHR. While tamsulosin treatment and tilting lowered BP and increased conductance in all vascular beds, effect size was not influenced by pretreatment with quercetin. Prolonged treatment with quercetin, in either prehypertensive SHR or adult SHR with established hypertension did not lower BP. CONCLUSIONS AND IMPLICATIONS: Cumulatively, these data demonstrate that quercetin does not amplify haemodynamic effects of tamsulosin or tilting in vivo in rats and has no effect on BP development in SHR.

Renal inflammatory markers during the onset of hypertension in spontaneously hypertensive rats.

Early blockade of the renin-angiotensin system is successful in delaying the development of hypertension in spontaneously hypertensive rats (SHRs) and ameliorating organ damage by inhibition of the inflammatory response. In this study, we investigated the role of the angiotensin II type 1 receptor (AT1R) in the early renal inflammatory response in SHR. Blood pressure development and renal inflammatory markers were measured in 4-, 8- and 12-week-old SHR and age-matched Wistar Kyoto (WKY) rats. Separate groups of SHRs were transiently treated with the AT1R blocker losartan between 4 and 8 weeks of age. Urinary excretion of the renal injury markers osteopontin and neutrophil gelatinase-associated lipocalin increased in young SHR. Further, renal expression of inflammatory genes was also increased in young SHR. Losartan inhibited the increase of these inflammatory markers. In contrast, gene expression of the renal injury marker and T-cell inducer kidney injury molecule-1 (KIM-1) was reduced in 4-week-old SHR when compared with WKY. Similarly, the T-cell marker CD3 was significantly decreased in 4-week-old SHR. These effects were not antagonized by AT1R blockade. This study confirms the presence of an early renal inflammatory response in SHR that can be blocked by AT1R antagonism. In addition, it demonstrates that KIM-1 does not behave as a pure kidney injury marker in young SHR, but may reflect kidney maturation.

Irreversible renal damage after transient renin-angiotensin system stimulation: involvement of an AT1-receptor mediated immune response.

Transient activation of the renin-angiotensin system (RAS) induces irreversible renal damage causing sustained elevation in blood pressure (BP) in Cyp1a1-Ren2 transgenic rats. In our current study we hypothesized that activation of the AT1-receptor (AT1R) leads to a T-cell response causing irreversible impairment of renal function and hypertension. Cyp1a1-Ren2 rats harbor a construct for activation of the RAS by indole-3-carbinol (I3C). Rats were fed a I3C diet between 4-8 weeks of age to induce hypertension. Next, I3C was withdrawn and rats were followed-up for another 12 weeks. Additional groups received losartan (20 mg/kg/day) or hydralazine (100 mg/kg/day) treatment between 4-8 weeks. Rats were placed for 24h in metabolic cages before determining BP at week 8, 12 and 20. At these ages, subsets of animals were sacrificed and the presence of kidney T-cell subpopulations was investigated by immunohistochemistry and molecular marker analysis. The development of sustained hypertension was completely prevented by losartan, whereas hydralazine only caused a partial decrease in BP. Markers of renal damage: KIM-1 and osteopontin were highly expressed in urine and kidney samples of I3C-treated rats, even until 20 weeks of age. Additionally, renal expression of regulatory-T cells (Tregs) was highly increased in I3C-treated rats, whereas the expression of T-helper 1 (Th1) cells demonstrated a strong decrease. Losartan prevented these effects completely, whereas hydralazine was unable to affect these changes. In young Cyp1a1-Ren2 rats AT1R activation leads to induction of an immune response, causing a shift from Th1-cells to Tregs, contributing to the development of irreversible renal damage and hypertension.

Fadrozole reverses cardiac fibrosis in spontaneously hypertensive heart failure rats: discordant enantioselectivity versus reduction of plasma aldosterone.

Reversal of cardiac fibrosis is a major determinant of the salutary effects of mineralocorticoid receptor antagonists in heart failure. Recently, R-fadrozole was coined as an aldosterone biosynthesis inhibitor, offering an appealing alternative to mineralocorticoid receptor antagonists to block aldosterone action. The present study aimed to evaluate the effects of R- and S-fadrozole on plasma aldosterone and urinary aldosterone excretion rate and to compare their effectiveness vs. the mineralocorticoid receptor antagonist potassium canrenoate to reverse established cardiac fibrosis. Male lean spontaneously hypertensive heart failure (SHHF) rats (40 wk) were treated for 8 wk by sc infusions of low (0.24 mg/kg.d) or high (1.2 mg/kg.d) doses of R- or S-fadrozole or by potassium canrenoate via drinking water (7.5 mg/kg.d). At the high dose, plasma aldosterone levels were decreased similarly by R- and S-fadrozole, whereas urinary aldosterone excretion rate was reduced only by S-fadrozole. In contrast, whereas at the high dose, R-fadrozole effectively reversed preexistent left ventricular interstitial fibrosis by 50% (vs. 42% for canrenoate), S-fadrozole was devoid of an antifibrotic effect. The low doses of the fadrozole enantiomers did not change cardiac fibrosis or plasma aldosterone but similarly reduced urinary aldosterone excretion rate. In conclusion, R-fadrozole may possess considerable therapeutic merit because of its potent antifibrotic actions in the heart. However, the observed discordance between the aldosterone-lowering and antifibrotic effects of the fadrozole enantiomers raises some doubt about the mechanism by which R-fadrozole diminishes cardiac collagen and about the generality of the concept of lowering aldosterone levels to treat the diseased heart.

Intrapericardial delivery enhances cardiac effects of sotalol and atenolol.

Targeting drugs to the heart by intrapericardial (i.p.c.) delivery may be a promising strategy to obtain higher drug efficiencies with lesser side effects. We examined whether i.p.c. delivery of sotalol and atenolol in rats offers advantages over intravenous (i.v.) application. Following sustained IPC infusion of sotalol or atenolol, pericardial fluid levels exceeded plasma levels 97 and 134 times respectively (P < 0.01) resulting in 3.8 and 4.7 times higher overall left ventricular tissue drug levels (P < 0.05). In a second experiment, the effects of the i.p.c. or i.v. beta-blocker infusions on nitroprusside-induced tachycardia were studied in conscious rats. For both drugs, i.p.c. infusion of 0.03 mg/kg.h produced similar antitachycardiac effects as the 1 mg/kg.h i.v. dose. In a third set of studies, dP/dt max challenged by dobutamine infusion was assessed to study ventricular contractile function after i.v. and i.p.c. sotalol in anesthetized rats. i.p.c. sotalol infusion attenuated the dobutamine response curve to a greater extent than i.v. (P < 0.01). In conclusion, i.p.c. infusion of sotalol and atenolol results in high cardiac tissue concentrations with low systemic drug levels. Similar antitachycardiac effects can be obtained at a 10- to 30-fold lower dose compared with i.v. delivery. Also, depression of ventricular contractility is acquired at a substantially lower i.p.c. sotalol dose. Thus, beta-blocking properties of sotalol and atenolol can be greatly enhanced by applying them i.p.c.

Role of the Rhoa/Rho kinase system in flow-related remodeling of rat mesenteric small arteries in vivo.

In small arteries, a chronic blood flow reduction leads to inward hypotrophic remodeling, while a chronic blood flow elevation induces outward hypertrophic remodeling. The RhoA/Rho kinase system was shown to be modulated by shear stress, and to be involved in other kinds of vascular remodeling. The aim of this study was to investigate the role of RhoA/Rho kinase in flow-related small artery remodeling. Rat mesenteric small arteries were subjected to flow-modifying surgery. After 1, 2, 4, 16, and 32 days, the animals were sacrificed and small arteries were harvested. Messenger RNA was isolated and amplified. Using cDNA microarray analysis, the differential expression of >14,000 genes was analyzed, part of which was confirmed by RT-PCR. In vivo treatment with fasudil (3 mg/kg/day s.c.) was used to test the effect of Rho kinase inhibition. The main findings are that: (1) blood flow alteration modified the expression of approximately 5% of the genes by >2-fold, (2) flow reduction downregulated many RhoA-related cytoskeletal markers of smooth muscle cell phenotype, (3) many RhoA-related genes were rapidly (<1 day) regulated and (4) fasudil treatment potentiated the inward hypotrophic remodeling in response to chronically reduced flow. These results indicate the importance of the RhoA/Rho kinase system in flow-related small artery remodeling.

Functional and structural postglomerular alterations in the kidney of prehypertensive spontaneously hypertensive rats.

The kidney plays a major role in the development of hypertension. Following the Borst-Guyton theory of an altered set-point for fluid and electrolyte homeostasis we aim to investigate functional and structural renal parameter during the development of hypertension. Therefore we focus on counter current exchange related factors. We compared 4 and 8 weeks old Wistar Kyoto rats (WKY) and spontaneously hypertensive rats (SHR) concerning basic renal parameters as creatinine and phosphorus clearance and urinary osmolality. Mean arterial pressure (MAP) was measured intra-arterially. Vasa recta were investigated using immunohistochemistry for alpha-smooth-muscle actin (ASMA) and plastification for geometric analyses. Blood pressure was not yet significantly elevated in SHR at 4 weeks but at 8 weeks it was higher in SHR (116+/-7 vs. 102+/-4 mm Hg; p<0.01). Kidney weight/body weight ratio was lower in SHR at both ages. In 4 weeks old SHR, phosphorus clearance and urinary osmolality were decreased compared to WKY [0.02+/-0.01 vs. 0.05+/-0.02 (ml/min* 100 g BW) p < 0.03; 14.2+/-2.2 vs. 18.9+/-2.9 (osmol/kg*24 h urine) p < 0.051 indicating reduced tubular reabsorption. At 8 weeks phosphorus clearance and urinary osmolality were comparable to WKY. alpha-Actin was found in vasa recta in a 4-times higher degree in SHR with a predominant location in the outer medulla. Radii of vasa recta in the outer medulla decreased during development. In plastificated sections vasa recta of SHR revealed sphincter-like pattern. Functional and structural alterations related to the counter current exchanger are already evident in prehypertensive SHR. During development of hypertension both factors get adapted to higher blood pressure level. Sphincter-like structures in vasa recta suggest contractility of pericytes/vascular smooth muscle cells (vSMC). As these were just seen in SHR that might allude to a higher potential to contract. We conclude that differences in postglomerular structure and function may contribute to the development of hypertension in SHR.

Pharmacokinetic advantage of intrapericardially applied substances in the rat.

Intrapericardial application of therapeutic agents may open perspectives for target-directed therapy of the diseased heart. This study was performed to investigate whether intrapericardial drug application is beneficial from a pharmacokinetic point of view. Male Wistar rats were provided with intrapericardial and intravascular catheters for substance administration and sampling. Intrapericardial bolus injections of fluorescent macromolecules [fluorescein isothiocyanate (FITC)-rat IgG, molecular weight about 155 kDa; Texas Red rat serum albumin, mol. wt. 67 kDa; Texas Red fibroblast growth factor (FGF), mol. wt. 18 kDa; and FITC heparin, mean mol. wt. 18 kDa] resulted in substance concentrations in pericardial fluid that exceeded those in plasma, for several hours. Pericardial fluid volumes of catheter-instrumented rats, derived from (initial) central compartment volumes, ranged between 0.5 and 0.9 ml/kg. After chronic (7 days) intrapericardial infusions with osmotic minipumps, pericardial fluid/plasma concentration ratios (local advantages) were 7 to 10 for the fluorescent proteins and >30 for FITC-heparin. This can be explained by the low substance clearances in pericardial fluid compared with plasma. Local advantages of the small substances cortisol (mol. wt. = 362.5) and a carbonic acid derivative thereof (mol. wt. = 348) were 14 and 420. Intrapericardial infusion of (125)I-FGF-2 yielded 8 times higher cardiac tissue levels than systemic infusion, whereas (125)I-FGF-2 was found in the entire heart. Pharmacokinetic profiles of intrapericardially applied substances are such that desired local drug concentrations can be obtained at lower dosages, whereas systemic concentrations remain low (thus reducing the potential risk of peripheral side effects). Therefore, intrapericardial application of therapeutic agents provides a promising strategy for site-specific treatment of heart or coronary diseases.