Hypertensive TGR Research Articles

Decreased sympathetic nerve activity in young hypertensive rats reared by normotensive mothers

AimsEarly postnatal development can be significantly compromised by changes in factors provided by the mother, leading to increased vulnerability to hypertension in her offspring. TGR(mRen-2)27 (TGR) mothers, characterised by an overactivated renin-angiotensin system, exhibit altered ion composition in their breast milk. Therefore, we aimed to analyse the impact of cross-fostering on cardiovascular parameters in hypertensive TGR and normotensive Hannover Sprague-Dawley (HanSD) offspring. Materials and methodsWe measured cardiovascular parameters in 5- to 10-week-old male offspring by telemetry. The expression of proteins related to vascular function was assessed by western blotting in the aortic samples obtained from 6- to 12-week-old male offspring. Plasma renin activity and plasma angiotensin II (Ang II) levels were evaluated by radioimmunoassay (RIA). Key findingsThe development of hypertension was in TGR accompanied by increased low-to-high frequency ratio (LF/HF; a marker of sympathovagal balance; 0.51 ± 0.16 in week 10). Furthermore, TGR exhibited increased aortic expression of mineralocorticoid receptor (MR; p < 0.05) and transforming growth factor beta type 1 (TGF-β1; p = 0.002) compared to HanSD offspring. Fostering significantly decreased sympathovagal balance (0.23 ± 0.10 in week 10) and, transiently, plasma Ang II levels and MR expression in TGR offspring reared by HanSD mothers. SignificanceThese findings highlight the importance of understanding the complex interplay between early life experiences, maternal factors, and later cardiovascular function. Understanding the mechanisms behind the observed effects may help to identify potential interventions to prevent the development of hypertension later in life.

Low Dose Curcumin Administered in Hyaluronic Acid-Based Nanocapsules Induces Hypotensive Effect in Hypertensive Rats.

BackgroundVascular drug delivery becomes a promising direction in the development of novel therapeutic strategies in the treatment of cardiovascular pathologies, such as hypertension. However, targeted delivery of hydrophobic substances, with poor bioavailability, remains a challenge. Here, we described the hypotensive effects of a low dose of curcumin delivered to the vascular wall using hyaluronic acid-based nanocapsules.MethodsThe group of hypertensive TGR(m-Ren2)27 rats, was administrated respectively with the vehicle, curcumin solution or curcumin delivered using hyaluronic acid-based nanocapsules (HyC12-Cur), for 7 days each, maintaining the wash-out period between treatments. Arterial blood pressure (systolic - SBP, diastolic – DBP) and heart rate (HR) were monitored continuously using a telemetry system (Data Science International), and Mean Arterial Pressure (MAP) was calculated from SBP and DBP.ResultsIn hypertensive rats, a low dose of curcumin (4.5 mg/kg) administrated in HyC12-Cur for 7 days resulted in a gradual inhibition of SBP, DBP and MAP increase without an effect on HR. At the end of HyC12-Cur – based treatment changes in SBP, DBP and MAP amounted to −2.0±0.8 mmHg, −3.9±0.7 mmHg and −3.3±0.7 mmHg, respectively. In contrast, the administration of a curcumin solution (4.5 mg/kg) did not result in a significant hypotensive effect and the animals constantly developed hypertension. Vascular delivery of capsules with curcumin was confirmed using newly developed fluorine-rich nanocapsules (HyFC10-PFOB) with a shell based on a HA derivative and similar size as HyC12-Cur. HyFC10-PFOB gave fluorine signals in rat aortas analyzed ex vivo with a 19F NMR technique after a single intragastric administration.ConclusionThese results suggest that nanocapsules based on hyaluronic acid, the ubiquitous glycosaminoglycan of the extracellular matrix and an integral part of endothelial glycocalyx, may represent a suitable approach to deliver hydrophobic, poorly bioavailable compounds, to the vascular wall.

OS 05-03 DECREASED RHO-KINASE-BASED CALCIUM SENSITIZATION IN HYPERTENSIVE REN-2 TRANSGENIC RATS (TGR)

Objective: Comparing BP response to Ca channel opener BAY K8644 prior and after Rho kinase inhibition we demonstrated lowered calcium sensitization in spontaneously hypertensive rats (SHR), although BP response of intact SHR to Rho kinase inhibitor fasudil was enhanced (Behuliak et al. J Hypertens 31:2025,2013). The aim of the present study was to evaluate calcium sensitization in hypertensive TGR and their normotensive HanSD controls using the same methods. We also tried to elucidate the interference of baroreflex-activated sympathetic activity with BP response to fasudil. Design and Method: Blood pressure and heart rate responses to increasing doses of BAY K8644 (opener of L type voltage-dependent calcium channels) was determined in conscious heterozygous TGR rats (subjected to acute RAS and SNS blockade). BP response was recorded prior and after Rho kinase inhibition by fasudil and calcium sensitization was estimated from the difference between both BP response curves. Furthermore, BP-lowering effects of acute administration of increasing fasudil doses were measured in conscious intact rats and in rats subjected to combined blockade of RAS, SNS and NOS in which baroreflex efficiency was suppressed. Results: Using the BAY K8644-based approach we demonstrated moderately attenuated calcium sensitization in hypertensive TGR but enhanced acute BP response to fasudil in intact TGR compared to HanSD rats. This was accompanied by greater heart rate increase in HanSD than in TGR, indicating attenuated baroreflex efficiency in hypertensive TGR. If baroreflex operation was minimized by ganglionic blockade, fasudil-induced BP fall was substantially augmented in HanSD but unchanged in TGR. Conclusions: Calcium sensitization is attenuated in hypertensive TGR, this change being evident only in the absence of sympathetic nervous system which compensates BP changes elicited by acute Rho kinase inhibition better in normotensive than in hypertensive animals. Supported by grant AZV 15–25396A (MH CR)

PP.31.05] ALTERED CALCIUM SENSITIZATION IN HYPERTENSIVE REN-2 TRANSGENIC RATS

Objective: A comparison of BP response to Ca channel opener BAY K8644 prior and after Rho kinase inhibition indicated decreased calcium sensitization in spontaneously hypertensive rats (SHR), although blood pressure (BP) response of intact SHR to Rho kinase inhibitor fasudil was enhanced (Behuliak et al. J Hypertens 31: 2025, 2013). The aim of the present study was to evaluate calcium sensitization in hypertensive transgenic rats with murine Ren-2 gene (TGR) and their normotensive HanSD controls using the same methods. We also tried to elucidate the interference of baroreflex-activated sympathetic activity with BP response to fasudil. Design and method: Blood pressure and heart rate responses to increasing doses BAY K8644 (opener of L type voltage-dependent calcium channels) was determined in conscious rats (subjected to acute RAS and SNS blockade). BP response was recorded prior and after Rho kinase inhibition by fasudil and calcium sensitization was estimated from the difference between both BP response curves. Furthermore, BP-lowering effects of acute administration of increasing fasudil doses were measured in conscious intact rats as well as in rats subjected to combined blockade of RAS, SNS and NOS in which baroreflex efficiency was suppressed. Results: Using the above BAY K8644-based approach we demonstrated moderate attenuation of calcium sensitization in hypertensive TGR. We also observed enhanced acute BP response to fasudil in intact TGR compared to HanSD rats. This different BP response was accompanied by more pronounced heart rate increase in HanSD than in TGR, indicating attenuated baroreflex efficiency in hypertensive TGR. If baroreflex operation was minimized by ganglionic blockade in rats subjected to combined RAS, SNS and NO synthase blockade, fasudil-induced BP fall was manifold augmented in HanSD but unchanged in TGR, suggesting the importance of baroreflex-activated sympathetic activity in compensation of fasudil-induced BP changes. Conclusions: Calcium sensitization is attenuated in hypertensive TGR compared to normotensive controls, but this difference can be seen only in the absence of operating sympathetic nervous system which compensates BP changes elicited by acute Rho kinase inhibition much better in normotensive than in hypertensive animals. Partially supported by grant AZV 15–25396A (MH CR)

PP.14.07

Objective: The recently developed combination of an Angiotensin Receptor blocker (ARB) and a Neprilysin Inhibitor (NEPi) has beneficial effects on clinical progression and mortality of patients with heart failure as compared to enalapril. However, since NEP also degrades endothelin-1 (ET-1), ARNI may cause side effects by increasing the levels of ET-1. Indeed, we recently observed in hypertensive heterozygote TGR(mREN2)27 rats that a low but not a high (0.1 vs. 1.0 mg/kg.day) dose of the NEPi thiorphan reduced blood pressure and cardiac hypertrophy on top of the ARB irbesartan. This was due to the fact that the high dose increased ET-1, upregulated constrictor vascular ET-1 type B receptors and induced an increase in renal sodium–hydrogen exchanger 3 protein abundance. In the present study, we studied the effects of the low thiorphan dose on top of irbesartan in diabetic TGR(mREN2)27 rats. Design and method: Heterozygote TGR(mREN2)27 rats were made diabetic with streptozotocin for 5 or 12 weeks. Rats were treated in the final 3 weeks with vehicle, irbesartan (15 mg/kg.day; IRB) or IRB plus the NEPi thiorphan (0.1 mg/kg.day; ARNI). Haemodynamics were measured by telemetry in the 5 week diabetic animals. In the 12 week diabetic animals vascular reactivity was determined in isolated mesenteric arteries, renal Na+ transporters were analysed by immunoblotting, and plasma and urine were collected for biochemical analysis. Results: Baseline mean arterial blood pressure (MAP) was 156.8 ± 5.4 mmHg. IRB and ARNI lowered MAP identically over the 3-week period, a maximum reduction of ∼50 mmHg being reached around day 7. Heart weight/tibia length ratio was reduced after treatment with ARNI only. Proteinuria and albuminuria were observed from 8 weeks of diabetes onwards and proteinuria was significantly reduced by ARNI treatment only. Urinary volume and plasma and urinary creatinine did not change. No ET-1 rises were observed, vascular reactivity was not influenced, and the pattern of kidney sodium transporters was not affected by ARNI or IRB treatment. Conclusions: ARNI reduces cardiac hypertrophy and proteinuria in diabetic TGR(mREN2)27 rats independently of blood pressure.

Abstract 507: Dual AT1 Receptor/Neprilysin (NEP) Inhibition (ARNI) vs. AT1 Receptor Blockade in TGR(mRen2)27 Rats: The More NEP Inhibition The Better?

Objective: Neprilysin inhibitors (NEPi) prevent the breakdown of natriuretic peptides, promoting vasodilation and natriuresis. However, they also increase angiotensin and endothelin-1 (ET1). This study compared the combination of an AT1 receptor antagonist (irbesartan, IRB) ± a low or a high dose of the NEPi thiorphan in renin-overexpressing, hypertensive TGR(mREN2)27 rats. Methods: TGR(mREN2)27 rats were treated for three weeks with vehicle, IRB (15 mg/kg.day) or IRB + thiorphan (0.1 or 1.0 mg/kg.day; TH0.1 and TH1.0). Hemodynamics were evaluated by telemetry, and vascular reactivity was determined in isolated mesenteric arteries (Mulvany myograph). Results: Baseline mean arterial blood pressure (MAP) was 168±3 mmHg. All treatments lowered MAP by ≈50 mmHg around day 4. After 7 days, MAP started to increase during treatment with IRB or IRB+TH1.0 (to 141±10 mmHg and 133±10 mmHg, respectively, on day 21), while MAP in rats treated with IRB+TH0.1 remained low at 104±5 mmHg on day 21. Heart weight/body weight ratio, cardiac ANP expression and myocyte size decreased only in the IRB+TH0.1 group. Plasma ET1 was increased only by TH1.0 versus IRB alone, and this increase was accompanied by an increase in renal sodium-hydrogen exchanger 3 (NHE3) protein expression: ET1-induced constriction was reduced by IRB+TH0.1 only. Vascular ET B R expression levels and studies with the ET1 type B receptor (ET B R) antagonist BQ788 revealed that this reduction was most likely due to ET B R upregulation. Conclusion: TH0.1 enhanced the blood pressure-lowering effects of irbesartan and diminished cardiac hypertrophy. Higher doses of thiorphan resulted in significant ET1 rises and renal NHE3 upregulation, thereby increasing blood pressure and sodium reabsorption. The simultaneously occurring upregulation of vasodilatory ET B R was insufficient to overcome this effect. Clearly therefore, too much NEPi on top of AT1 receptor antagonism might be harmful.

Renal effects of long-term darbepoetin alpha treatment in hypertensive TGR(mRen2)27 rats

Erytropoietin (EPO) has cytoprotective and angiogenic properties and has a beneficial effect in ischaemic conditions. Since the development of renal interstitial abnormalities are often associated with ischaemia, we studied the effects of the long-acting EPO analogue darbepoetin alpha (DA) on kidney damage in TGR(mRen2)27 (Ren2) rats. Ren2 rats were randomised to DA or vehicle (VEH) or to DA + angiotensin converting enzyme inhibitor (ACEi) or VEH + ACEi. Sprague Dawley (SD) rats served as controls. Blood pressure was measured weekly and 24-h urine was collected to measure proteinuria. Blood samples were collected for creatinine and haematocrit. Kidneys were studied for inflammation and pre-fibrosis. Renal mRNA expression was studied for EPO, EPO-receptor, collagen-3α1 and kidney injury molecule-1 (KIM-1). DA had no effect on SBP, serum creatinine and proteinuria. Interstitial and glomerular α-SMA expression was significantly increased in Ren2. ACEi but not DA improved the increased renal inflammatory and pro-fibrotic profile in Ren2 rats. DA on top of ACEi further reduced glomerular α-SMA and KIM-1 expression. Long-term DA treatment has no beneficial effects on renal structural and functional changes in TGR(mRen2)27 rats in the time frame studied and the dose provided.

Expressions of per1 clock gene and genes of signaling peptides vasopressin, vasoactive intestinal peptide, and oxytocin in the suprachiasmatic and paraventricular nuclei of hypertensive TGR[mREN2]27 rats.

Hypertensive rats with multiple extra copies of the renin gene (TGR) exert an inverted circadian blood pressure (BP) profile. We investigated whether circadian oscillations in the hypothalamic suprachiasmatic nucleus (SCN), a main circadian oscillator, and the paraventricular nucleus (PVN), involved in BP control, are influenced in TGR rats. The expression of the clock gene per1, a marker of circadian timing, was measured in the SCN and PVN. Moreover, the expression of genes encoding vasopressin (AVP), vasoactive intestinal peptide (VIP) in the SCN, and AVP and oxytocin (OXT) in the PVN were studied by in situ hybridization. Expression of the per1 gene showed a distinct circadian rhythm in both the SCN and PVN with no differences observed between the TGR and control Sprague–Dawley (SD) rats. The expression of avp in the SCN was rhythmic in both strains and moderately higher in TGR than in SD rats while no significant changes were found in the PVN. The expression of vip in the SCN and oxt in the PVN did not differ between both strains. Our results may indicate that changes occurring downstream to the SCN are responsible for the development of the inverted BP rhythm in TGR hypertensive rats.

Influence of up-regulated renin—angiotensin system on the exploration, anxiety-related behavior and object recognition

The renin-angiotensin system (RAS) plays an important role in the development of hypertension and has serious consequences on behaviour. The aim of our study was to investigate the effect of hypertension, induced by up-regulated RAS, on the exploration, anxiety-related behaviour and object recognition in laboratory rats. In the experiment, 12 weeks old normotensive Sprague-Dawley (SD) and hypertensive TGR(mREN2)27 (TGR) male rats with up-regulated RAS were used. In the open-field test, the TGR rats were less active in ambulating, rearing and sniffing and more active in self-grooming and urinating than SD ones. In the elevated plus-maze test, the TGR rats showed lower frequency of total arm entries, closed arm entries and higher frequency of defecation than in controls. In the emergence test, TGR rats did not show significant differences. In the novel object recognition task, the TGR rats spent less time with exploration of both familiar and unfamiliar objects but preferred the novel object over the familiar one and exhibited higher percentage of the total exploring time spent with novel object exploration than SD rats. Our results indicate that the TGR rats are less actively exploring, show some modifications of emotional/anxiety-related behavior and exhibited better recognition abilities.

Effect of rhythmic melatonin administration on clock gene expression in the suprachiasmatic nucleus and the heart of hypertensive TGR(mRen2)27 rats

Plasma melatonin concentrations in non-dipping patients show a blunted daily rhythm. Melatonin has a capacity to improve disturbances in biological rhythms. Hypertensive TGR(mRen2)27 (TGR) rats with an upregulated renin-angiotensin system and inverted blood pressure profile were used to elucidate whether melatonin is able to influence the control of blood pressure. Melatonin was administered in drinking water to normotensive Sprague-Dawley (SD) and hypertensive TGR rats during the dark phase of the light: dark cycle 12: 12 for 4 weeks. The effect of melatonin on blood pressure was monitored, and the expression of clock genes per2 and bmal1 and melatonin receptor MT1 in the suprachiasmatic nucleus (SCN) and the heart was measured by real time polymerase chain reaction during a 24-h cycle. The administration of melatonin did not influence clock gene expression in the SCN but its effect on clock gene expression in the heart was phase dependent in both SD and TGR rats. Melatonin administration did not decrease the expression of melatonin receptors in the SCN and the heart. Melatonin did not decrease blood pressure in TGR rats but influenced the peripheral oscillator in the heart independently of the SCN. A modified function of molecular circadian oscillators in the heart can interfere with anticipation and disturb the adaptation of this organ to pressure overload.

Endocrine Rhythms and Expression of Selected Genes in the Brain, Stellate Ganglia, and Adrenals of Hypertensive TGR Rats

Hypertensive TGR(mRen2)27 (TGR) rats represent a strain with genetically upregulated renin-angiotensin-aldosterone system. Simultaneously with development of hypertension, a daily profile in blood pressure (BP) inverts and in mature TGR rats BP is higher during the lighttime (L) than the darktime (D). Physiological mechanisms of inverted BP rhythm generation are not understood. In our study we determined circadian profiles of plasma hormones related to BP control (aldosterone, corticosterone, melatonin, prolactin) in TGR and control Sprague-Dawley (SD) rats over 24 h and expression of genes encoding catecholamine synthesizing enzymes, tyrosine hydroxylase (TH), dopamine-beta-hydroxylase (DBH), and phenylethanolamine-N-methyltransferase (PNMT) in adrenals and stellate ganglia. Plasma levels of corticosterone and aldosterone were higher in TGR than SD rats but acrophases of their rhythms were not changed. Darktime peak of prolactin in TGR rats was decreased in comparison with SD animals and pineal melatonin levels started to rise earlier in TGR than in SD rats. In adrenals we found upregulated expression of TH, DBH, and PNMT mRNA at the beginning of the lighttime in TGR compared to SD rats. Expression of TH and DBH in stellate ganglia was not different in TGR rats in comparison with SD, but PNMT expression was higher during L compared to D in TGR rats. We hypothesize that upregulated adrenal medulla functioning in the morning and disturbed communication between circadian oscillators and mechanisms involved in BP control can explain the reversed BP profile in TGR rats.

Omega-3 polyunsaturated fatty acid supplementation reduces hypertension in TGR(mRen-2)27 rats

To establish the effect of dietary ω-3 PUFA on angiotensin II (ANG II)-mediated hypertension, male TGR (mRen-2)27 (Ren-2) rats (animals with high ANG II activity) were maintained on a diet either deficient or sufficient in ω-3 PUFA from conception. Half the animals on each diet were treated with the angiotensin-converting enzyme inhibitor, perindopril, from birth. Ren-2 rats fed the ω-3 PUFA deficient diet were significantly more hypertensive than those fed the ω-3 PUFA sufficient diet. Perindopril reduced the blood pressure of both ω-3 PUFA-deficient and ω-3 PUFA-sufficient diet-fed Ren-2 rats. Body weight, body fat and plasma leptin were reduced by perindopril treatment but not affected by ω-3 PUFA supply. Given that the elevated blood pressure of the Ren-2 rat is mediated by ANG II, the data suggest that ω-3 PUFA may reduce hypertension via the renin–angiotensin system.

Effect of upregulated renin–angiotensin system on per2 and bmal1 gene expression in brain structures involved in blood pressure control in TGR(mREN-2)27 rats

Circadian system regulates rhythms with 24 h period including those occurring in the cardiovascular system. Inverted blood pressure profile was demonstrated in hypertensive TGR(mREN-2)27 (TGR) rats with upregulated renin-angiotensin system. To depict structures involved in the generation of the inverted pattern of blood pressure in TGR rats, we analyzed daily expression of clock genes per2 and bmal1 in the brain areas involved in the regulation of the blood pressure. Heterozygous male TGR and control rats were synchronized to the light:dark cycle 12:12 and blood samples were taken in 4 h intervals within 24 h cycle. The levels of the plasma renin activity were increased in TGR rats in comparison with controls. Brain nuclei were isolated by dissection from frozen sections. The clock gene expression was determined in the hypothalamic paraventricular and dorsomedial nuclei, dorsal vagal motor nucleus, caudal ventrolateral medulla, nucleus ambiguus, area postrema, and anteroventral third ventricle. Daily pattern of per2 expression was rhythmic in most of the nuclei studied with its highest levels at the beginning of the nighttime in both groups of rats. Expression of bmal1 peaked at the beginning of the day. We found robust differences in the clock gene expression between the TGR and control rats in the area postrema. TGR rats exerted changes in the clock gene expression in the nucleus ambiguus which receives direct innervation from the area postrema. The area postrema seems to play a key role in the transmission of signals from the periphery to the CNS.

Rhythmic clock gene expression in heart, kidney and some brain nuclei involved in blood pressure control in hypertensive TGR(mREN-2)27 rats

Hypertensive TGR(mREN-2)27 rats exerting inverted blood pressure (BP) profile were used to study clock gene expression in structures responsible for BP control. TGR and control Sprague Dawley male rats were synchronized to the light:dark cycle 12:12 with food and water ad libitum. Daily rhythm in per2, bmal1, clock and dbp expression in the suprachiasmatic nucleus (SCN), rostral ventrolateral medulla (RVLM), nucleus of the solitary tract (NTS), heart and kidney was determined in both groups. Sampling occurred in regular 4 h intervals when rats of both strains were 11-weeks-old. Blood pressure and relative heart weight were significantly elevated in TGR rats in comparison with control. Expression of bmal1 and clock was up regulated in SCN of TGR rats but daily rhythm in per2 and dbp expression was similar in both groups. Mesor of per2 expression in RVLM was significantly higher in TGR than in control rats. In NTS of TGR rats expression of per2 was phase delayed by 3.5 h in comparison with control and bmal1 did not exert rhythmic pattern. Our study provided the first evidence about modified function of central and peripheral circadian oscillators in TGR rats at the level of clock gene expression. Expression of clock genes exerted up regulation in SCN and RVLM and down regulation in NTS. Circadian oscillators in selected brain structures were influenced more than oscillators in the heart and kidney by additional renin gene. Interactions of RAS and circadian system probably contribute to the development of inverted BP profile in TGR rats.

Early-onset endothelin receptor blockade in hypertensive heterozygous Ren-2 rats

Male heterozygous Ren-2 transgenic rats and Hannover Sprague–Dawley rats fed a normal or high-salt diet were either untreated or treated with the nonselective receptor ETA/ETB receptor blocker bosentan or the selective ETA receptor blocker, ABT-627, known as atrasentan. Survival rate was partly increased by bosentan and fully normalized by atrasentan. Bosentan did not significantly influence the course of hypertension in TGR, whereas atrasentan significantly decreased BP on both diets. Atrasentan substantially reduced proteinuria, cardiac hypertrophy, glomerulosclerosis and left ventricular ET-1 tissue concentration on both diets. Our data indicate that ETA receptor blockade is superior to nonselective blockade in attenuating hypertension, end-organ damage and improving survival rate.

αV Integrins Are Necessary for Eutrophic Inward Remodeling of Small Arteries in Hypertension

Human essential hypertension is characterized by eutrophic remodeling of small arteries, with little evidence of hypertrophy. Likewise, vessels of young hypertensive TGR(mRen2)27 animals have undergone similar structural alterations. The role of integrins in resistance arteries of TGR(mRen2)27 during the eutrophic-remodeling process was examined as blood pressure rose. Initially, 8 alpha and 3 beta integrins were identified and levels of expression investigated using RT-PCR. As pressure increased and remodeling advanced, integrin expression profiles revealed that only alphaV was significantly raised. In conjunction, we confirmed elevated integrin alphaV protein levels in TGR(mRen2)27 rat arteries and localization to the media using immunofluorescence. beta1 and beta3, but not beta5 integrin subunits were coprecipitated with integrin alphaV and are implicated in the eutrophic remodeling process. Administration of a peptide antagonist of alphaVbeta3 abolished remodeling but enhanced growth, indicating that hypertrophy supervened as a response to hypertension-induced increases in wall stress. We have established that the only upregulated integrin, the alphaV subunit of integrin alphaVbeta3, has a crucial role in the hypertensive remodeling process of TGR(mRen2)27 rat resistance arteries. During hypertensive remodeling, functions of specific alphaVbeta3-extracellular matrix interactions are likely to allow vascular smooth muscle cell-length autoregulation, which includes a migratory process, to maintain a narrowed lumen after a prolonged constricted state.

Inverse Blood Pressure Rhythm of Transgenic Hypertensive TGR(mREN2)27 Rats: Role of Norepinephrine and Expression of Tyrosine‐Hydroxylase and Reuptake1‐Transporter

Transgenic hypertensive TGR(mREN2)27 rats (TGR) exhibit an inverse circadian blood pressure profile from the age of 8 to 9 wk. To investigate the role of the sympathetic nervous system in this pathological blood pressure rhythm, we examined postnatal changes in catecholamine concentration, expression of tyrosine‐hydroxylase (TH), and norepinephrine (NE) reuptake1‐transporter (NET) in the heart, adrenal glands, and hypothalamus of non‐hypertensive TGR at an age of 4 wk and of hypertensive TGR at an age of 10 wk and compared these to normotensive, age‐matched Sprague‐Dawley rats. Rats were kept under synchronized light:dark (LD) conditions of 12:12 h. Blood pressure and heart rate were monitored by radiotelemetry, catecholamines by high performance liquid chromatography, expression of TH and NET (mRNA) by RT‐PCR, and TH protein by Western blots. In normotensive 4 wk‐old Sprague‐Dawley rats, cardiac NE concentrations were circadian phase‐dependent with lower values at ZT12.5, with no differences observed, in 10‐wk‐old animals. At both ages however, sympathetic tone was higher during the dark phase, as shown by a higher turnover of NE. This observation confirms earlier data, which indicate that the endogenous amine concentration may not mirror its turnover rate. TGR at either age had lower cardiac NE as well as lower TH expression and did not display a circadian phase‐dependency. The increased cardiac NE turnover rate in the dark phase in non‐hypertensive TGR was lost in hypertensive rats. Both cardiac NE concentrations and TH expression decreased with age in both strains. In adrenal glands, NE and epinephrine (E) were not circadian phase‐dependent in both strains but increased with age. NE concentrations in the hypothalamus were neither circadian phase‐dependent nor different in both strains and at both ages. However, sympathetic tone of NE in the hypothalamus, as indicated by the turnover rate, was greater during the dark phase in both strains at an age of 10 wk. Expression of TH and NET were greatly reduced in adrenal glands when compared to Sprague‐Dawley rats; whereas, expression of TH in the hypothalamus was significantly increased in hypertensive TGR. These data indicate that the transgene in TGR leads to an increased central stimulation of the sympathetic nervous system and to a consecutive down‐regulation in the peripheral organs. It is of interest that rhythmicity in the studied parameters was lost in hypertensive TGR, except in the turnover of NE in the hypothalamus. We concluded that the data on key mechanisms of regulation of the sympathetic system in TGR cannot explain the inverse blood pressure rhythm observed in this transgenic rat strain.

Effects of sodium restriction and cyclooxygenase-2 inhibition on the course of hypertension, proteinuria and cardiac hypertrophy in Ren-2 transgenic rats

The present study was performed to evaluate the effects of sodium intake and of chronic cyclooxygenase-2 (COX-2) inhibition on systolic blood pressure (SBP) in heterozygous male transgenic rats harboring the mouse Ren-2 renin gene (TGR) and in transgene-negative normotensive Hannover Sprague-Dawley (HanSD). Twenty-eight days old TGR and HanSD were randomly assigned to groups fed either normal salt (NS) or low sodium (LS) diets. COX-2 blockade was achieved with NS-398 (1 mg x kg(-1).day(-1) in drinking water). During an experimental period of 26 days, SBP was repeatedly measured by tail plethysmography in conscious animals. We found that the LS diet prevented the development of hypertension in TGR and did not change SBP in HanSD. Low sodium intake also prevented proteinuria and cardiac hypertrophy in TGR. On the other hand, irrespective of sodium intake chronic COX-2 inhibition did not alter the course of SBP in either TGR or HanSD. The present data indicate that TGR exhibit an important salt-sensitive component in the developmental phase of hypertension. They also suggest that systemic COX-2-derived prostaglandins do not act as vasodilatory counterregulatory agents in TGR in which an exaggerated vascular responsiveness to angiotensin II is assumed as the pathophysiological mechanism in the development of hypertension.

Increased expression of IL-6 and LIF in the hypertrophied left ventricle of TGR(mRen2)27 and SHR rats

Cytokines from the interleukin-6 (IL-6) family have been reported to play an important synergistic role with angiotensin II in the development of pathological cardiac hypertrophy. Whether their expression pattern changes in vivo, in an angiotensin I-dependent hypertrophied myocardium has not been reported. In this study, we addressed that issue using two animal models of angiotensin II-dependent cardiac hypertrophy. Heterozygous transgenic TGR(mRen2)27 (TGR) with an overactive cardiac renin angiotensin system and the closely related spontaneously hypertensive rats (SHR) were compared to their respective control rats. The mRNA levels of IL-6, leukemia inhibitory factor (LIF), ciliary neurotrophic factor (CNTF) and cardiotrophin-1 (CT-1) as well as their receptor subunits, glycoprotein 130 (gp130), IL-6 receptor (IL-6R), LIFR, and CNTFR, were measured by semi-quantitative RT-PCR. The protein levels of IL-6, LIF and CT-1 were investigated by western blot. TGR and SHR both displayed significant over expression of mRNA and protein levels for IL-6 and LIF. In TGR, the increased level of LIF was accompanied by a decrease in mRNA levels for LIFR and CNTFR. In SHR, a higher level of mRNA IL-6R was observed. By contrast, the mRNA and protein levels for CT-1 and the mRNA level for gp130 did not vary in these two models. These findings suggest that IL-6 and LIF, but not CT-1, contribute to angiotensin II-dependent left ventricular hypertrophy in the two hypertensive rat models, TGR(mRen2)27 and SHR.

Transgenic TGR(mREN2)27 Rats as a Model for Disturbed Circadian Organization at the Level of the Brain, the Heart, and the Kidneys

In transgenic hypertensive TGR(mREN2)27 rats (TGR) harboring the murine Ren‐2 gene an inverse 24h blood pressure (BP) profile was described in relation to a normal pattern in heart rate (HR) and motility (MA), normotensive Sprague‐Dawley rats (SDR) were used as controls. Transgenic rats as an animal model of human secondary hypertension (non‐dipper) was studied in detail at different levels: (1) Radiotelemetry was applied to document gross circadian rhythms/rhythm disturbances in cardiovascular functions, MA and body temperature under normal LD conditions, under DD and after a light pulse. (2) Signal transduction of the overexpressed renin‐angiotensin in TGR was studied by determation of AT1‐receptors in kidney glomeruli together with kidney functions. (3) Expression of key processes involved in increased sympathetic regulation in TGR, mRNAs, the tyrosine‐hydroxylase (TH) and norepinephrine (NE) reuptake1‐carrier were determined. (4) In the SCN mRNA of c‐fos and c‐jun were determined under LD and after light pulse. (5) In primary cultures of pinealocytes the effects of adrenergic agonists and antagonists were evaluated on second messenger (cAMP, cGMP) accumulation and melatonin release. The results of these studies clearly demonstrate that the additional mouse renin genin in TGR greatly affected not only the renin‐angiotensin‐system and led—as expected—to an increased BP in this rat but also disturbed circadian rhythms from the BP pattern down to the level of hormones, processes of signal transduction, and expression of transcription factors and clock genes. In conclusion, the expression of a single additional gene is able to disturb the circadian system of an animal in a highly complex way. These findings are of importance for chronobiologic as well as pharmacologic research.