TGR Rats Research Articles

Soluble guanylate cyclase stimulator as an emerging therapeutic option in the management of arrhythmias in heart failure rat model

Abstract Funding Acknowledgements Type of funding sources: Foundation. Main funding source(s): This research was supported by VEGA grant no. 2/0002/20 and 2/0006/23, the Slovak Research and Development Agency under contract no. 21-0410, European Regional Development Fund: ITMS2014+: 313011AVG3. This research was also partially supported by the Ministry of Health of the Czech Republic within the project for the development of the research organization [grant number 00023001 (IKEM)—institutional support] and also supported by the project National Institute for Research of Metabolic and Cardiovascular Diseases (Program EXCELES, Project No. LX22NPO5104)—Funded by the European Union—Next Generation EU Heart failure (HF) is a complex clinical syndrome characterized by a decrease in the efficiency of the heart pump, leading to a decline in hemodynamic status and cardiac remodelling, that can contribute to the formation of a substrate for arrhythmias. Antiarrhythmic drug therapy is still suboptimal and current therapies are not efficacious enough. The purpose of the current study was therefore to examine the efficiency of nitric oxide (NO)/soluble guanylyl cyclase (sGC)/cyclic guanosine monophosphate (cGMP) stimulator. We used as an experimental model male hypertensive Ren-2 transgenic rats (TGR, n = 16), normotensive Hannover Sprague–Dawley rats (HSD, n = 17), and a rat model of HF surgically induced by aortocaval fistula (ACF) in rats of both strains (n = 27). After ACF induction were rats treated by sGC stimulator BAY41-8543 (3 mg/kg/day for 30 weeks) alone or combined with angiotensin-converting enzyme inhibitor (ACEi) Trandolapril (0.25 mg/kg/day for 30 weeks). Age of the animals at the end of the experiment was 40 weeks. Left ventricle tissue and plasma samples were used for further analyses. Treatment by sGC stimulator significantly decreased rats' mortality, and systolic blood pressure, and significantly increased levels of antioxidant enzyme superoxide dismutase 1, matrix metalloproteinase 2, total and phosphorylated connexin 43 as well as protein kinase C epsilon, implicated in remodelling of extracellular matrix and intercellular communication, in the left ventricle of TGR rats. Application of sGC stimulator slightly normalized elevated collagen deposition and hydroxyproline content in the left ventricular tissue of TGR rats. Unfortunately, we did not observe any effect of the sGC stimulator in the TGR ACF group. These results support the hypothesis that sGC stimulators might represent a class of drugs suitable for combating heart failure with partially anti-arrhythmic potential, but further studies are needed.

Transgenic angiotensin-converting enzyme 2 overexpression in the rat vasculature protects kidneys from ageing-induced injury

Chronic kidney disease is one of the leading causes of morbidity and mortality especially among the aged population. A decline in kidney function with ageing comparable to ageing-related processes in human kidneys has also been described in Sprague-Dawley (SD) rats. The renin-angiotensin-system (RAS) plays a pivotal role in the pathophysiology of cardiovascular and kidney disease and is a successful therapeutic target. The discovery of angiotensin-(1-7) (Ang(1-7)), mainly produced by angiotensin-converting enzyme 2 (ACE2), and its receptor MAS offered a new view on the RAS. This ACE2/Ang(1-7)/MAS axis counteracts most deleterious actions of the RAS in the kidney. In order to evaluate if activation of this axis has a protective effect in ageing-induced kidney disease we generated a transgenic rat model (TGR(SM22hACE2)) overexpressing human ACE2 in vascular smooth muscle cells. These animals showed a specific transgene expression pattern and increased ACE2 activity in the kidney. Telemetric recording of cardiovascular parameters and evaluation of kidney function by histology and urine analysis revealed no alterations in blood pressure regulation and basal kidney function in young transgenic rats when compared to young SD rats. However, with ageing, SD rats developed a decline in kidney function characterized by severe albuminuria which was significantly less pronounced in TGR(SM22hACE2) rats. Concomitantly, we detected lower mRNA expression levels of kidney damage markers in aged transgenic animals. Thus, our results indicate that vascular ACE2-overexpression protects the kidney against ageing-induced decline in kidney function, supporting the kidney-protective role of the ACE2/Ang(1-7)/MAS axis.

PS-B02-6: CHANGES OF THE EXTRACELLULAR MATRIX AND INTERCELLULAR COMMUNICATION IN THE RAT HEARTS WITH VOLUME-PRESSURE OVERLOAD AND MODULATION BY CARDIOPROTECTIVE DRUGS

Objective: Pathogenesis of heart failure (HF) and hypertension with a focus on extracellular matrix (ECM) and communication in the heart is still a current scientific topic that calls for new findings. Design and method: We used as experimental model normotensive (HSD) and hypertensive (TGR) rats with aortocaval fistula (ACF) supplemented with an ACE inhibitor (ACEi)(15weeks, 6 mg/L) and an AT1 receptor blocker (ARB)(15weeks, 200 mg/L). At the end of the experiments, serum and myocardial tissue samples of the right and left ventricles were taken for biometric, biochemical, gene, protein and microscopic analyzes. Results: We observed an increase in the cardiac weight index after ACF, left and right ventricles in HSD and TGR rats. ACEi and ARB have normalized this condition to a greater extent. Due to ACF, serum concentrations of ANP and TBARS were increased in the blood serum of both strains. ACF increased hydroxyproline levels in right ventricular levels in HSD and TGR rats and left ventricular TBARS in TGR rats. In addition, ACF increased mRNA collagenI, and collagenI protein expression in the left and right ventricles of HSD rats. In contrast, ACF reduced Cx43 and pCx43 protein levels in the left and right ventricles of both strains, SMAD2 expression in the right ventricle in HSD and TGR rats. ACF also suppressed MMP-2 protein expression in both cardiac ventricles in HSD rats and PKCepsilon protein expression in both heart chambers of both strains. Hypertension caused a slight decrease in the protein expression of Cx43, pCx43 and PKCepsilon and an increase in the expression of PKCdelta in cardiac ventricles of both rat strain. Hypertension further increased and collagenI in TGR rats in the left and right ventricles of the heart. Microscopic analyzes showed that ACF induced cardiomyocyte hypertrophy as well as decreased activity of enzymes involved in ACF energy metabolism. An interesting finding was that the signaling pathways involved in cardiac remodeling were more normalized by the ARB, while proteins involved in intercellular communication by ACEi. Conclusions: Based on the results obtained from the experiment, it can be stated that the right ventricle is more significantly affected by ACF and the results of analyzes in TGR indicate adaptation, attenuated responses to the parameters we observed. Supported by: APVV-21-410, VEGA-2/0158/19.

Role of Endothelial Kinin B1 Receptor on the Membrane Potential of Transgenic Rat Aorta

The kinin receptors are classically involved in inflammation, pain and sepsis. The effects of the kinin B1 receptor agonist des-Arg9-bradykinin (DBK) and lipopolysaccharide (LPS) were investigated by comparing the membrane potential responses of aortic rings from transgenic rats overexpressing the kinin B1 receptor (B1R) in the endothelium (TGR(Tie2B1)) and Sprague Dawley (SD) rats. No difference in the resting membrane potential in the aorta's smooth muscle from the transgenic and SD rats was observed. The aorta rings from SD rats hyperpolarized only to LPS but not to DBK, whereas the aorta rings from TGR(Tie2B1) responded by the administration of both drugs. DBK and LPS responses were inhibited by the B1 receptor antagonist R715 and by iberiotoxin in both cases. Thapsigargin induced a hyperpolarization in the smooth muscle of SD rats that was not reversed by R715, but was reversed by iberiotoxin and this hyperpolarization was further augmented by DBK administration. These results show that the model of overexpression of vascular B1 receptors in the TGR(Tie2B1) rats represent a good model to study the role of functional B1 receptors in the absence of any pathological stimulus. The data also show that KCa channels are the final mediators of the hyperpolarizing responses to DBK and LPS. In addition, we suggest an interaction between the B1R and TLR4, since the hyperpolarization induced by LPS could be abolished in the presence of R715.

INHIBITION OF RAS COMPONENTS ATTENUATES PROGRESSION OF MYOCARDIAL REMODELING IN BOTH NORMOTENSIVE AND HYPERTENSIVE RATS IN EXPERIMENTAL HEART FAILURE

Objective: New findings in the pathogenesis of heart failure (HF) with a focus on extracellular matrix (ECM) and communication in the heart are still relevant for more effective treatment. We focused on signaling pathways involved in cardiac structural remodeling in HF due to pressure and volume overload, as well as on investigating the efficacy of substances with possible antifibrotic potential. Design and method: We used as experimental model 28-week-old hypertensive (TGR) and normotensive (HSD) rats with created aortocaval fistula (ACF), supplemented with ACE inhibitor (ACEi) (Trandolapril, 15 weeks, 6 mg/L) and AT1 receptor blocker (ARB) (Losartan, 15 weeks, 200 mg/L). Results: Echocardiography confirmed an increase in cardiac output and a decrease in ejection fraction in both strains of rats. BW was higher in TGR vs HSD and it was not affected by VO. ACF-induced cardiac volume overload, elevated markers of heart failure and oxidative damage, ANP, BNP-45, and TBARS, were elevated in the blood serum of both strains. ACF induced profibrotic signaling by increasing TGF-, and mRNA collagen I, and collagen I protein expression in the left and right ventricles of HSD rats. In TGR rats ACF decreased TGF- and SMAD2 protein expression in the right ventricle similar to HSD rats, in which ACF also suppressed MMP-2 protein expression. Heart failure affected protein kinase signaling by reducing the protein expression of PKC, PKG, and in part, PKC in both chambers of the heart of both strains. ACF decreased the protein expression of Cx43 and its phosphorylated form, which is significantly proarrhythmic. Hypertension in TGR rats increased mRNA TGF- expression and collagen I, but decreased PKC and PKC protein expression and increased PKC. Conclusions: A notable finding was that the signaling pathways involved in cardiac remodeling were more normalized by the ARB, while proteins involved in intercellular communication, ACEi. The results of analyzes of both experiments indicate attenuated responses of the signaling pathways monitored by us in the heart of hypertensive strains reflecting adaptation to primarily induced pathophysiology. Scientific Grant Agency of the Ministry of Education, Science, Research and Sport of the Slovak Republic VEGA 2/0158/19, 2/0002/20

AT1 receptor blocker, but not an ACE inhibitor, prevents kidneys from hypoperfusion during congestive heart failure in normotensive and hypertensive rats

To provide novel insights into the pathogenesis of heart failure-induced renal dysfunction, we compared the effects of ACE inhibitor (ACEi) and AT1 receptor blocker (ARB) on systemic and kidney hemodynamics during heart failure in normotensive HanSD and hypertensive transgenic (TGR) rats. High-output heart failure was induced by creating an aorto-caval fistula (ACF). After five weeks, rats were either left untreated or treatment with ACEi or ARB was started for 15 weeks. Subsequently, echocardiographic, renal hemodynamic and biochemical measurements were assessed. Untreated ACF rats with ACF displayed significantly reduced renal blood flow (RBF) (HanSD: 8.9 ± 1.0 vs. 4.7 ± 1.6; TGR: 10.2 ± 1.9 vs. 5.9 ± 1.2 ml/min, both P < .001), ACEi had no major RBF effect, whereas ARB completely restored RBF (HanSD: 5.6 ± 1.1 vs. 9.0 ± 1.5; TGR: 7.0 ± 1.2 vs. 10.9 ± 1.9 ml/min, both P < .001). RBF reduction in untreated and ACEi-treated rats was accompanied by renal hypoxia as measured by renal lactate dehydrogenase activity, which was ameliorated with ARB treatment (HanSD: 40 ± 4 vs. 42 ± 3 vs. 29 ± 5; TGR: 88 ± 4 vs. 76 ± 4 vs. 58 ± 4 milliunits/mL, all P < .01). Unlike improvement seen in ARB-treated rats, ACE inhibition didn’t affect urinary nitrates compared to untreated ACF TGR rats (50 ± 14 vs. 22 ± 13 vs. 30 ± 13 μmol/mmol Cr, both P < .05). ARB was more effective than ACEi in reducing elevated renal oxidative stress following ACF placement. A marker of ACEi efficacy, the angiotensin I/angiotensin II ratio, was more than ten times lower in renal tissue than in plasma. Our study shows that ARB treatment, in contrast to ACEi administration, prevents renal hypoperfusion and hypoxia in ACF rats with concomitant improvement in NO bioavailability and oxidative stress reduction. The inability of ACE inhibition to improve renal hypoperfusion in ACF rats may result from incomplete intrarenal RAS suppression in the face of depleted compensatory mechanisms.

Intravitreal injection of mesenchymal stem cells evokes retinal vascular damage in rats.

The aim of this study is to investigate the vascular outcome after intravitreal mesenchymal stem cell (MSC) administration in rats without or with damage to the neurovascular unit [transgenic (TGR) rats]. Male Sprague-Dawley (SD) and TGR rats received an intravitreal injection of 2 × 104 rat bone marrow-derived MSCs (BMSCs) or human adipose-derived stem cells (ASCs) at postnatal d 30. After 4 wk, vasculature, neuronal function, and gene expression in the retinas were evaluated using retinal morphometry, electroretinography, immunofluorescence, Western blot, and quantitative PCR. Intravitreal administration of rat BMSCs and human ASCs in both SD and TGR eyes induced cataract, loss of pericytes, and increased formation of acellular capillaries. BMSCs remained in the vitreous cavity and did not migrate into the retinas. Intravitreal administration of BMSCs impacted retinal neuronal function in neither SD nor TGR rats. Retinal glial activation, elevation of IL-1β, C3, arginase 1, and heat shock protein 90 were detected in BMSC-injected SD rats. Intravitreal administration of MSCs induces cataract, retinal vasoregression, activation of retinal glial cells, and inflammatory response in rat eyes.-Huang, H., Kolibabka, M., Eshwaran, R., Chatterjee, A., Schlotterer, A., Willer, H., Bieback, K., Hammes, H.-P., Feng, Y. Intravitreal injection of mesenchymal stem cells evokes retinal vascular damage in rats.

P3513Sex-related differences in the outcome of congestive heart failure: study on rats treated with ACEi alone or combined with soluble epoxide hydrolase inhibitor

Abstract Introduction In search for new therapeutic measures of congestive heart failure (CHF) attention focused on the role of epoxyeicosatrienoic acids (EETs), cytochrome P450-dependent epoxygenase pathway metabolites of arachidonic acid, with antihypertensive and organ-protective actions. The EETs are rapidly broken-down by soluble epoxide hydrolase (sEH). Blocking sEH and increasing tissue EETs bioavailability had antihypertensive and cardio- and renoprotective effects. The studies of the biological mechanisms underlying the sex-related differences in the CHF and in the responses to new pharmacological measures are missing. The rat model in which CHF is induced by volume overload by creation of the aorto-caval fistula (ACF) is recommended for preclinical studies by American Heart Association. It has been noticed that the hypertensive rat transgenic for the mouse Ren-2 renin gene (TGR) presents a unique angiotensin II-dependent model of hypertension. We found that male ACF TGR displayed tissue deficiency of EETs, and increasing intrarenal EETs levels by pharmacological blockade of sEH attenuated the progression of CHF in male ACF TGR. However, we did not examine if this effect occurs also in female ACF TGR. Purpose The aim was to establish if sex-related differences, if present, are demonstrable with standard treatment with ACEi alone and with the combined treatment with ACEi and sEHi. Methods Male and female TGR rats were randomly assigned either to ACF procedure or to sham-operation. One week after the procedure rats were divided into the 8 experimental groups (sham-operated male TGR + placebo, ACF male TGR + placebo, ACF male TGR + ACEi, ACF male TGR + ACEi + sEHi, sham-operated female TGR + placebo, ACF female TGR + placebo, ACF female TGR + ACEi, ACF female TGR + ACEi + sEHi). C-AUCB was used as an sEHi and trandolapril as ACEi, both in drinking water in doses previously tested. The follow-up period was 50 weeks and the primary end-point was death from any cause. Results All sham-operated male and female TGR survived until the end of experiment. All untreated male ACF TGR animals died by week 20. In contrast, untreated female ACF survived in the rate of 32%. The treatment with ACEi improved survival rate similary in male as well as female ACF TGR (74% and 65%). The combined treatment with ACEi and sEHi worsened the survival in male ACF TGR as compared with ACF TGR + ACEi (38%). In contrast, the combined treatment with ACEi and sEHi in female ACF TGR significantly improved the course and the final survival rate (84%) as compared with female ACF TGR + ACEi alone. Picture 1 Conclusions The study suggests that in CHF individuals in whom hypertension and increased RAS activity run in parallel, the patients' sex is the co-determinant of CHF progression. In particular it can influence the effectiveness of the therapeutic measures applied to slow it down. Therefore, in pre- and clinical studies the sex-related differences should be seriously considered. Acknowledgement/Funding Ministry of Health of the Czech Republic grant no. 17- 28220A; Grant Agency of Charles University, project number 32218

Reduced anxiety-like behavior in transgenic rats with chronically overproduction of angiotensin-(1–7): Role of the Mas receptor

Angiotensin-(1–7) [Ang-(1–7)], a counterregulatory peptide of the renin-angiotensin system (RAS), exerts its cardiovascular and renal functions through the G-protein-coupled receptor Mas. More recently, Ang-(1–7) has also been implicated in the control of emotional states related to fear and anxiety. Here, we tested the hypothesis that transgenic rats overexpressesing Ang-(1–7) (TGR) show reduced anxiety-like behavior in two distinct animals models, the Elevated Plus Maze (EPM) and Vogel Conflict Test (VCT). Sprague-Dawley rats (SDs) were used as controls. In addition, we also verified whether this phenotype depend on activation of the Mas receptor. In line with our hypothesis, TGR rats showed an increase in the percentage of time and entries in the open arms of the EPM. There was also an increase in the number of punished licks in VCT. These phenotypes were reversed by ICV injection of the Mas receptor antagonist, A779, but not by the AT2 and MrgD receptor antagonist, PD123319. These results suggest that chronic elevation of Ang-(1–7) levels results in a phenotype characterized by reduced anxiety-like behavior, possibly due to higher activation of the Mas receptor. Therefore, facilitation of the Ang-(1–7)/Mas receptor signaling may be further investigated as an additional strategy for the treatment of anxiety-related disorders.

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)

The brain renin-angiotensin system plays a crucial role in regulating body weight in diet-induced obesity in rats.

Reduced weight gain after treatment with AT1 receptor antagonists may involve a brain-related mechanism. Here, we investigated the role of the brain renin-angiotensin system on weight regulation and food behaviour, with or without additional treatment with telmisartan. Transgenic rats with a brain-specific deficiency in angiotensinogen (TGR(ASrAOGEN)) and the corresponding wild-type, Sprague Dawley (SD) rats were fed (3months) with a high-calorie cafeteria diet (CD) or standard chow. SD and TGR(ASrAOGEN) rats on the CD diet were also treated with telmisartan (8mg·kg(-1) ·d(-1) , 3months). Compared with SD rats, TGR(ASrAOGEN) rats (i) had lower weights during chow feeding, (ii) did not become obese during CD feeding, (iii) had normal baseline leptin plasma concentrations independent of the feeding regimen, whereas plasma leptin of SD rats was increased due to CD, (iv) showed a reduced energy intake, (v) had a higher, strain-dependent energy expenditure, which is additionally enhanced during CD feeding, (vi) had enhanced mRNA levels of pro-opiomelanocortin and (vii) showed improved glucose control. Weight gain and energy intake in rats fed the CD diet were markedly reduced by telmisartan in SD rats but only to a minor extent in TGR(ASrAOGEN) rats. The brain renin-angiotensin system affects body weight regulation, feeding behaviour and metabolic disorders. When angiotensin II levels are low in brain, rats are protected from developing diet-induced obesity and obesity-related metabolic impairments. We further suggest that telmisartan at least partly lowers body weight via a CNS-driven mechanism.

Locus Coeruleus Dysfunction in Transgenic Rats with Low Brain Angiotensinogen.

Transgenic TGR(ASrAOGEN)680 (TGR) rats with specific downregulation of glial angiotensinogen (AOGEN) synthesis develop cardiovascular deficits, anxiety, altered response to stress, and depression. Here, we evaluated whether these deficits are associated with alteration of the integrity of the noradrenergic system originating from locus coeruleus (LC) neurons. Adult TGR rats were compared to control Sprague Dawley rats in terms of the following: tissue levels of transcripts encoding noradrenergic markers, tissue tyrosine hydroxylase (TH) protein level, in vivo TH activity, density of TH-containing fibers, behavioral response to novelty, locomotor activity, and polysomnography. TH expression was increased in the LC of TGR rats compared to controls. In LC terminal fields, there was an increase in density of TH-containing fibers in TGR rats that was associated with an elevation of in vivo TH activity. TGR rats also displayed locomotor hyperactivity in response to novelty. Moreover, polysomnographic studies indicated that daily paradoxical sleep duration was increased in TGR rats and that the paradoxical sleep rebound triggered by total sleep deprivation was blunted in these rats. Altogether, these results suggest that disruption of astroglial AOGEN synthesis leads to cardiovascular, cognitive, behavioral, and sleep disorders that might be partly due to LC dysfunction.

Angiotensin-(1-7) attenuates the anxiety and depression-like behaviors in transgenic rats with low brain angiotensinogen

Transgenic rats with low brain angiotensinogen, TGR(ASrAOGEN)680, expressing an antisense RNA against angiotensinogen in glial cells, provide an interesting tool to evaluate the role of brain angiotensins in different behavior responses. The present study was conducted to test the hypothesis that angiotensin-(1-7) [Ang-(1-7)] and serotonin can modulate anxiety and depression-related behaviors in the TGR(ASrAOGEN)680 rats. Therefore, the effect of acute intracerebroventricular administration of Ang-(1-7) and intraperitoneal administration of the selective serotonin reuptake inhibitor fluoxetine was evaluated in TGR(ASrAOGEN) rats subjected to the elevated plus maze (EPM) and forced swimming (FST) tests. Transgenic rats spent a lower percentage of time in the open arms of EPM and showed a significant increase in the immobility time in FST, indicating that a low angiotensinogen level in the brain leads to anxiety-like behavior accompanied by a depression-like state. Administration of both, Ang-(1-7) and fluoxetine reversed the anxiety- and depressive-like behavior of transgenic rats with low brain angiotensinogen, suggesting that this may be, at least in part, related to a decreased level of Ang-(1-7) and serotonin in the brain of these animals.

Chronic hyperglycemia inhibits vasoregression in a transgenic model of retinal degeneration

Vasoregression characterizes diabetic retinopathy in animal models and in humans. We have recently demonstrated that vasoregression is earlier initiated in a rat model of ciliopathy-induced retinal neurodegeneration (TGR rat). The aim was to assess the balance between vasoregressive effects of chronic hyperglycemia and photoreceptor degeneration on adult vascular remodelling. The retinas were analyzed at 4 and 9 months after streptozotocin-induced diabetes. Neurodegeneration was determined by quantitation of cell numbers and retinal layer thickness. Vasoregression was assessed by quantitative retinal morphometry in retinal digest preparations. Retinal VEGF levels were measured by ELISA. Glial activation, expression and location of HSP27 and phosphorylated HSP27 were evaluated by immunofluorescence staining. Unexpectedly, the numbers of acellular capillaries were reduced at both time points and led to fewer intraretinal microvascular abnormalities in late stage diabetic TGR. Concomitantly, inner nuclear layers (INLs) in diabetic TGR rats were protected from cell loss at both time points. Consequently, glial activation was reduced, but VEGF level was increased in diabetic TGR retinas. Expressions of HSP27 were upregulated in glia cells in the preserved INL of diabetic TGR. Chronic hyperglycemia preserves the microvasculature in the retinal model of neurodegeneration. Cell preservation in the retinal INL was associated with protective gene regulation. Together, these data indicate that diabetes can induce vasoprotection, in which retinal glia can play a particular role.

Evidence that kinin B2 receptor expression is upregulated by endothelial overexpression of B1 receptors

Bradykinin (BK) and des-Arg9-bradykinin (DBK) of kallikrein-kinin system exert its effects mediated by the B2 (B2R) and B1 (B1R) receptors, respectively. It was already shown that the deletion of kinin B1R or of B2R induces upregulation of the remaining receptor subtype [10,12,16,28,36]. However studies on overexpression of B1R or B2R in transgenic animals have supported the importance of the overexpressed receptor but the expression of another receptor subtype has not been determined [17,19,33]. Previous study described a marked vasodilatation and increased susceptibility to endotoxic shock which was associated with increased mortality in response to DBK in thoracic aorta from transgenic rat overexpressing the kinin B1R (TGR(Tie2B1)) exclusively in the endothelium. In another study, mice overexpressing B1R in multiple tissues were shown to present high susceptibility to inflammation and to lipopolysaccharide-induced endotoxic shock. Therefore the role of B2R was investigated in the thoracic aorta isolated from TGR(Tie2B1) rats overexpressing the B1R exclusively in the vascular endothelium. Our findings provided evidence for highly increased expression level of the B2R in the transgenic rats. It was reported that under endotoxic shock, these rats exhibited exaggerated hypotension, bradycardia and mortality. It can be suggested that the high mortality during the pathogenesis of endotoxic shock provoked in the transgenic TGR(Tie2B1) rats could be due to the enhanced expression of B2R associated with the overexpression of the B1R.

Increased circulating angiotensin-(1–7) protects white adipose tissue against development of a proinflammatory state stimulated by a high-fat diet

IntroductionThe aim of the present study was to evaluate the effect of a transgenic-induced chronic increase of Ang-(1–7) on the expression of inflammatory markers in adipose tissue and the metabolic profile in rats treated with high-fat diet. Research design and methodsTransgenic rats expressing an Ang-(1–7)-producing fusion protein (TGR L-3292) and Sprague Dawley (SD) control rats 4weeks old were treated for 8weeks with a high-fat diet. Food intake and body weight were measured once a week. Glucose-tolerance and insulin sensitivity tests were performed one week before the sacrifice. At the end of the experiment plasma lipid concentrations were measured in TGR and SD rats. Adipose tissue were weighted and corrected by the body weight. Proinflammatory markers in adipose tissue were analyzed using Western-blotting, real time-PCR and immunohistochemistry. ResultsHigh-fat diet TGR rats presented increased HDL cholesterol levels and decreased abdominal fat mass, without changes in food intake. In addition, rats with increased Ang-(1–7) levels had lower body weight. Molecular analysis revealed decreased IL-1β and COX-2 in adipose tissue. ConclusionsTaken together, these results show that chronic high circulating angiotensin-(1–7) levels protect against metabolic stress induced by a high-fat diet decreasing the proinflammatory profile of adipose tissue.

Cardiovascular responses evoked by activation or blockade of GABAA receptors in the hypothalamic PVN are attenuated in transgenic rats with low brain angiotensinogen

Previous evidence indicates that a balance between inhibitory gabaergic and excitatory angiotensinergic factors in the PVN is important for cardiovascular control. We investigated the cardiovascular response evoked from activation or blockade of GABAA receptors in the paraventricular nucleus (PVN), in transgenic rats with low brain angiotensinogen [TGR(ASrAOGEN)]. Brain Ang II and Ang-(1–7) levels were also determined. In functional experiments, TGR(ASrAOGEN) and Sprague–Dawley rats (SD, control) were anesthetized with urethane and blood pressure (BP), heart rate (HR) and renal sympathetic nerve activity (RSNA) were recorded. Brain Ang II and Ang-(1–7) levels were largely reduced in TGR(ASrAOGEN) compared with SD rats. Inhibition of PVN neurons with the GABAA agonist, muscimol (1nmol/100nL), resulted in an attenuated fall in all cardiovascular variables in TGR(ASrAOGEN) compared with SD rats. This difference was particularly pronounced in HR (TGR Mus −23±6bpm vs. −77±9bpm SD Mus; P<0.05) and RSNA (TGR −3±10% vs.−29±8% SD; P<0.05). Furthermore, the sympathetic response evoked by blockade of GABAA receptors in the PVN of TGR(ASrAOGEN) was also largely suppressed. The present data indicate that the sympathetic outflow mediated by PVN neurons under basal conditions is suppressed in TGR(ASrAOGEN) rats corroborating the functional significance of brain angiotensin production in the central regulation of sympathetic output to the cardiovascular system.

A new transgenic rat model overexpressing the angiotensin II type 2 receptor provides evidence for inhibition of cell proliferation in the outer adrenal cortex

This study aimed to elucidate the role of the AT(2) receptor (AT(2)R), which is expressed and upregulated in the adrenal zona glomerulosa (ZG) under conditions of increased aldosterone production. We developed a novel transgenic rat (TGR; TGRCXmAT(2)R) that overexpresses the AT(2)R in the adrenal gland, heart, kidney, brain, skeletal muscle, testes, lung, spleen, aorta, and vein. As a consequence the total angiotensin II (Ang II) binding sites increased 7.8-fold in the kidney, 25-fold in the heart, and twofold in the adrenals. The AT(2)R number amounted to 82-98% of total Ang II binding sites. In the ZG of TGRCXmAT(2)R, the AT(2)R density was elevated threefold relative to wild-type (WT) littermates, whereas AT(1)R density remained unchanged. TGRCXmAT(2)R rats were viable and exhibited normal reproduction, blood pressure, and kidney function. Notably, a slightly but significantly reduced body weight and a moderate increase in plasma urea were observed. With respect to adrenal function, 24-h urinary and plasma aldosterone concentrations were unaffected in TGRCXmAT(2)R at baseline. Three and 14 days of Ang II infusion (300 ng·min(-1)·kg(-1)) increased plasma aldosterone levels in WT and in TGR. These changes were completely abolished by the AT(1)R blocker losartan. Of note, glomerulosa cell proliferation, as indicated by the number of Ki-67-positive glomerulosa cells, was stimulated by Ang II in TGR and WT rats; however, this increase was significantly attenuated in TGR overexpressing the AT(2)R. In conclusion, AT(2)R in the adrenal ZG inhibits Ang II-induced cell proliferation but has no obvious lasting effect on the regulation of the aldosterone production at the investigated stages.

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.

The influence of leptin on trabecular architecture and marrow adiposity in GH-deficient rats

The relationship between the degree of GH deficiency and impaired bone integrity is not simple and may be influenced by related endocrine variables. To test the hypothesis that elevated adiposity and hyperleptinaemia are contributory factors, we quantified femoral trabecular organisation in two models of GH deficiency with divergent degrees of adiposity - the moderately GH-deficient/hyperleptinaemic transgenic growth retarded (Tgr) rat and the profoundly GH-deficient/hypoleptinaemic dw/dw rat. Trabecular density (bone volume/total volume) and surface were reduced by 16% in dw/dw males, with a more fragmented trabecular lattice. This impairment was more pronounced in Tgr rats, with trabecular number and density further reduced (by an additional 21%) and relative surface (bone surface/bone volume), trabecular convexity (structural modal index) and fragmentation (pattern factor) increased. To establish whether the presence of obesity/hyperleptinaemia exacerbates bone impairment in GH deficiency, trabecular structure was assessed in dw/dw rats following diet-induced obesity (DIO). DIO had minimal effect on trabecular architecture, the increased concavity of trabecular surfaces being the only observable effect. Similarly, infusion of leptin into the tibial bone marrow cavity had no effect on trabecular organisation or tibial growth in wild-type rats. However, while this procedure also failed to affect trabecular architecture or osteoclast number in dw/dw rats, distal osteoblast surface was increased by 23%, marrow adipocyte number and epiphyseal plate width being reduced (by 40 and 5% respectively), without increasing caspase-3 immunoreactivity. These findings suggest that while leptin may directly inhibit adipocyte differentiation and favour osteoblast production, hyperleptinaemia makes only a minimal contribution to the impairment of bone structure in GH deficiency.