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Vitamin D deficiency is one of the most common nutritional deficiencies worldwide. Maternal vitamin D deficiency is associated with increased susceptibility to hypertension in offspring, but the reasons for this remain unknown. The aim of this study was to determine if parental vitamin D deficiency leads to altered DNA methylation in offspring that may relate to hypertension. Male and female Sprague-Dawley rats were fed a standard or vitamin D-depleted diet. After 10 wk, nonsibling rats were mated. The conceived pups received standard chow. We observed an increased systolic and diastolic blood pressure in the offspring from depleted parents (F1-depl). Genome-wide methylation analyses in offspring identified hypermethylation of the promoter region of the Pannexin-1 ( Panx1) gene in F1-depl rats. Panx1 encodes a hemichannel known to be involved in endothelial-dependent relaxation, and we demonstrated that in F1-depl rats the increase in blood pressure was associated with impaired endothelial relaxation of the large vessels, suggesting an underlying biological mechanism of increased blood pressure in children from parents with vitamin deficiency. Parental vitamin D deficiency is associated with epigenetic changes and increased blood pressure levels in offspring.

Diabetic nephropathy is still a common complication of type 2 diabetes mellitus (T2DM) and improvement of endothelial dysfunction (ED) and inhibition of reactive oxygen species (ROS) are considered important targets for new therapies. Recently, we developed a new class of compounds (Sul compounds) which inhibit mitochondrial ROS production. Here, we tested the therapeutic effects of Sul-121 on ED and kidney damage in experimental T2DM. Diabetic db/db and lean mice were implanted with osmotic pumps delivering Sul-121 (2.2 mg/kg/day) or vehicle from age 10 to 18 weeks. Albuminuria, blood pressure, endothelial mediated relaxation, renal histology, plasma creatinine, and H2O2 levels were assessed. Sul-121 prevented progression of albuminuria and attenuated kidney damage in db/db, as evidenced by lower glomerular fibronectin expression (~50%), decreased focal glomerular sclerosis score (~40%) and normalization of glomerular size and kidney weight. Further, Sul-121 restored endothelium mediated vasorelaxation through increased production of Nitric Oxide production and normalized plasma H2O2 levels. Sul-121 treatment in lean mice demonstrated no observable major side-effects, indicating that Sul-121 is well tolerated. Our data show that Sul-121 inhibits progression of diabetic kidney damage via a mechanism that involves restoration of endothelial function and attenuation of oxidative stress.

Metformin confers vascular benefits beyond glycemia control, possibly via pleiotropic effects on endothelial function. In type-1-diabetes-mellitus (T1DM-)patients metformin improved flow-mediated dilation but also increased prostaglandin(PG)-F2α, a known endothelial-contracting factor. To explain this paradoxical finding we hypothesized that metformin increased endothelial-vasodilator mediators (e.g. NO and EDHF) to an even larger extent. Spontaneously-hypertensive-rats (SHR) display impaired endothelium-dependent relaxation (EDR) involving contractile PGs. EDR was studied in isolated SHR aortas and the involvement of PGs, NO and EDHF assessed. 12-week metformin 300 mg/kg/day improved EDR by up-regulation of NO and particularly EDHF; it also reduced blood pressure and increased plasma sulphide levels (a proxy for H2S, a possible mediator of EDHF). These effects persisted in SHR with streptozotocin (STZ)-induced T1DM. Vildagliptin (10 mg/kg/day), targeting the incretin axis by increasing GLP-1, also reduced blood pressure and improved EDR in SHR aortas, mainly via the inhibition of contractile PGs, but not in STZ-SHR. Neither metformin nor vildagliptin altered blood glucose or HbA1c. In conclusion, metformin reduced blood pressure and improved EDR in SHR aorta via up-regulation of NO and particularly EDHF, an effect that was independent from glycemia control and maintained during T1DM. A comparison to vildagliptin did not support effects of metformin mediated by GLP-1.

Type 2 diabetes mellitus (T2DM) is associated with risk for chronic kidney disease (CKD), which is associated with a decrease in renal myogenic tone - part of renal autoregulatory mechanisms. Novel class of drugs used for the treatment of T2DM, dipeptidyl peptidase-4 (DPP-4) inhibitors, have protective effects on the cardiovascular system. A Zucker Diabetic Fatty (ZDF) rat is an animal model of T2DM that displays progressive nephropathy in which inflammation leads to initiation of renal fibrosis and CKD. We hypothesized that CKD in the ZDF rat is related to decrease in myogenic constriction (MC) of intrarenal arteries and that treatment with the DPP-4 inhibitor, vildagliptin, prevents such changes. Renal arteries isolated from 25 weeks old lean, ZDF and ZDF treated with vildagliptin (n=7 in each group) were transferred to an arteriograph to assess agonist and pressure induced contractile responses. Furthermore, blood glucose, proteinuria, focal glomerulosclerosis (FGS) and p22phox mRNA expression of renal tissue were measured. Compared to lean controls, ZDF had significantly increased plasma glucose and cholesterol levels, focal glomerulosclerosis and interstitial α-SMA expression, and urinary protein excretion. ZDF rats also had impaired MC of renal arteries and increased renal p22phox expression. Vildagliptin did not affect plasma glucose levels or proteinuria, but effectively decreased glomerulosclerosis and restored MC and p22phox expression to the levels found in lean rats. Based on these data, it can be suggested that vildagliptin treatment protects diabetic rats from the loss of renal vascular reactivity and the development of glomerulosclerosis perhaps secondary to a reduction in oxidative stress.

Background: Diabetes and particularly high blood glucose levels are implicated in neurodegeneration. One of the hallmarks of neurodegeneration is protein aggregation. We investigated the presence of protein aggregation in the frontal brain of Zucker diabetic fatty (ZDF) rats, an animal model for diabetes. Further, the effect of NaHS in suppressing protein aggregation in cultured brain slices from ZDF was assessed.Results: The levels of protein synthesis, protein/gene expression, autophagy and anti-oxidant defense were evaluated in ZDF and control (Lean) brains.Compared to Lean, ZDF brains displayed a significant increase in protein aggregates, p-tau, fibronectin expression and protein glycosylation. Increased phosphorylation of mTOR and S6 ribosomal protein in ZDF indicated higher protein synthesis, while the increase in ubiquitinated proteins and LC3-I in ZDF brains accompanied by lower LC3-II expression and LC3-II/LC3-I levels indicated the blockage of proteolytic pathways. CBS (cystathionine beta synthase) protein and mRNA expression and thiol group levels in ZDF brains were lower compared to Lean. ZDF brains show a higher level of reactive oxygen species. In vitro NaHS treatment normalized proteostasis while counteracting oxidative stress.Conclusion: Our data demonstrate increased protein synthesis and aggregation in the diabetic ZDF rat brain, which was reversible by NaHS treatment. This is the first report on the potential use of NaHS as a novel strategy against protein aggregation in diabetic brain.

Growth differentiation factor 15 (GDF15) is emerging as valuable biomarker in cardiovascular disease and diabetic kidney disease. Also, GDF15 represents an early response gene induced after tissue injury and studies performed in GDF15 knockout (KO) mice suggest that GDF15 plays a protective role after injury. In the current study, we investigated the role of GDF15 in the development of diabetic kidney damage in type 1 and type 2 models of diabetes. Renal damage was assessed in GDF15 KO mice and wild-type (WT) mice in streptozotocin type 1 and db/db type 2 diabetic models. Genetic deletion of GDF15 augmented tubular and interstitial damage in both models of diabetes, despite similar diabetic states in KO and WT mice. Increased tubular damage in KO animals was associated with increased glucosuria and polyuria in both type 1 and type 2 models of diabetes. In both models of diabetes, KO mice showed increased interstitial damage as indicated by increased α-smooth muscle actin staining and collagen type 1 expression. In contrast, glomerular damage was similarly elevated in diabetic KO and WT mice. In type 1 diabetes, GDF15 KO mice demonstrated increased expression of inflammatory markers. In type 2 diabetes, elevated levels of plasma creatinine indicated impaired kidney function in KO mice. GDF15 protects the renal interstitium and tubular compartment in experimental type 1 and 2 diabetes without affecting glomerular damage.

Introduction:Intact myogenic constriction plays a role in renal blood flow autoregulation and protection against pressure-related (renal) injury. However, to what extent alterations in renal artery myogenic constriction are involved in development of renal damage during aging is unknown. Therefore, we studied two strains of fawn-hooded rats, which differ in expression of hypertension and chronic renal failure.Methods:Ten-week-old fawn-hooded hypertensive (FHH) and fawn-hooded low blood pressure (FHL) rats were followed for SBP and proteinuria for 1 year. At 52 weeks of age, the kidney was removed and studied for focal glomerulosclerosis (FGS) and glomerular cross-sectional area, and myogenic constriction of isolated small renal arteries in a vessel perfusion set up. Renal myogenic constriction and FGS were additionally determined in 10-week-old fawn-hooded rats.Results:At young age, fawn-hooded rats did not differ in SBP, FGS, and urinary protein excretion, but renal artery myogenic constriction already was significantly smaller (approximate to 50%) in FHH compared with FHL rats. Aging in fawn-hooded rats was associated with increase in SBP and urinary protein excretion and loss of renal artery myogenic constriction. These changes occurred in both fawn-hooded strains, although that in FHH rats the onset of hypertension occurred earlier and the increase in proteinuria by far exceeded (>4 times) that observed in FHL rats, and came along with 5.5 times increase in FGS and 1.3 times increase in glomerular cross-sectional area and significantly less preserved renal artery myogenic constriction in FHH rats.Conclusion:Better preservation of renal myogenic constriction protects the kidney from age-related hypertensive renal damage in the fawn-hooded rat.

OBJECTIVE: Over the last two decades, the radial artery (RA) has become a routinely used conduit for coronary artery bypass graft surgery. One potential disadvantage of the radial artery is its higher susceptibility to vasospasm compared with other arterial grafts. We investigated whether adventitial dissection of the radial artery can reduce vasoconstriction and increase free blood flow.METHODS: Following harvesting, the adventitia of the radial artery was dissected using coronary scissors. Surplus distal radial artery segments (n = 35) with and without adventitial dissection of patients undergoing coronary artery bypass surgery were collected and pairwise assessment of vasoreactivity to potassium chloride, U46619 and acetylcholine was performed in organ bath experiments. Free blood flow was measured before and after adventitial dissection.RESULTS: Full curve and maximal vasoconstriction of the RA to potassium chloride (P = 0.015 and 0.001) and U46619 (P = 0.048 and 0.001) was significantly reduced after adventitial dissection compared with non-adventitial dissected radial arteries. Endothelium-dependent relaxation to acetylcholine of adventitial dissected radial arteries was significantly increased (P = 0.006) compared with non-adventitial dissected radial arteries. Maximal vasorelaxation to acetylcholine was significantly increased for adventitial dissected radial arteries compared with non-adventitial dissected radial arteries (P = 0.018). Free blood flow was significantly increased after adventitial dissection (P = 0.037).CONCLUSION: The adventitial dissected radial artery is less susceptible to vasoconstriction and more prone to vasorelaxation ex vivo and shows an increased free blood flow. Therefore, we suggest adventitial dissection of the radial artery graft to reduce vasospasm for arterial revascularization in coronary artery bypass surgery.

Transactivation of epidermal growth factor receptor (EGFR) signaling by G protein-coupled receptors has been implicated in several cardiovascular (CV) conditions, including hypertension, heart failure, and cardiac and vascular hypertrophy. However, the therapeutic potential of EGFR inhibition in these conditions is currently unknown. The main objective of the present study was to investigate cardiac, vascular, and renal effects of EGFR inhibition by 4-[4-[[(1R)-1-phenylethyl]amino]-7H-pyrrolo[2,3-d]pyrimidin-6-yl]phenol (PKI-166) in the hypertensive chronic kidney disease model. Rats underwent 5/6 nephrectomy (5/6Nx) and were treated with PKI-166, lisinopril or vehicle from week 6 after disease induction until week 12. Sham animals received either PKI-166 or vehicle. Treatment with PKI-166 did not affect the development of the characteristic renal features in 5/6Nx, including proteinuria, diminished creatinine clearance, and increased glomerulosclerosis, whereas these were attenuated by lisinopril. Despite absence of effects on progressive renal damage, PKI-166 attenuated the progression of hypertension and maintained cardiac function (left ventricle end-diastolic pressure) to a similar extent as lisinopril. Also, PKI-166 attenuated the increase in phosphorylated EGFR in the heart as induced by 5/6Nx. Moreover, PKI-166 and lisinopril restored the impaired contraction of isolated thoracic aortic rings to phenylephrine and angiotensin II and impaired myogenic constriction of small mesenteric arteries in 5/6Nx rats. Blockade of the EGFR displays a CV benefit independent of limiting the progression of renal injury. Our findings extend the evidence on EGFR signaling as a target in CV disorders.

Mazagova M, Buikema H, Landheer SW, Vavrinec P, van Buiten A, Henning RH, Deelman LE. Growth differentiation factor 15 impairs aortic contractile and relaxing function through altered caveolar signaling of the endothelium. Am J Physiol Heart Circ Physiol 304: H709-H718, 2013. First published December 21, 2012; doi:10.1152/ajpheart.00543.2012.-Growth differentiation factor 15 (GDF15) is an independent predictor of cardiovascular disease, and increased GDF15 levels have been associated with endothelial dysfunction in selected patients. We therefore investigated whether GDF15 modulates endothelial function in aortas of wild-type (WT) and GDF15 knockout (KO) mice. Vascular contractions to phenylephrine and relaxation to ACh were assessed in aortas obtained from healthy WT and GDF15 KO mice. The effects of GDF15 pretreatment and the involvement of ROS or caveolae were determined. Phenylephrine-induced contractions and ACh-mediated relaxations were similar in WT and GDF15 KO mice. Pretreatment with GDF15 inhibited contraction and relaxation in both groups. Inhibition of contraction by GDF15 was absent in denuded vessels or after blockade of nitric oxide (NO) synthase. Relaxation in WT mice was mediated mainly through NO and an unidentified endothelium-derived hyperpolarizin factor (EDHF), whereas GDF15 KO mice mainly used prostaglandins and EDHF. Pretreatment with GDF15 impaired relaxation in WT mice by decreasing NO; in GDF15 KO mice, this was mediated by decreased action of prostaglandins. Disruption of caveolae resulted in a similar inhibition of vascular responses as GDF15. ROS inhibition did not affect vascular function. In cultured endothelial cells, GDF15 pretreatment caused a dissociation between caveolin-1 and endothelial NO synthase. In conclusion, GDF15 impairs aortic contractile and relaxing function through an endothelium-dependent mechanism involving altered caveolar endothelial NO synthase signaling.

Objective: Since the cerebrovasculature likely plays a prominent role in the pathophysiology of eclampsia, we assessed the effects of low-dose endotoxin-induced experimental preeclampsia on the function and structure of rat posterior cerebral arteries (PCA) and mesenteric arteries (MA).Methods: Nonpregnant (NP) and pregnant (P) rats were infused with saline (NP-CTL, n = 9; P-CTL, n = 9) or low-dose endotoxin (NP-endotoxin, n = 9; P-endotoxin, n = 10). Myogenic activity, pressure of forced dilatation (FD) and structural properties were evaluated in PCA and MA.Results: PCA underwent FD between 125 and 150 mmHg in P-endotoxin (repeated measures ANOVA vs 75 mmHg; P Conclusion: Low-dose endotoxin-infusion during pregnancy, but not pregnancy alone, decreased the pressure of FD in PCA. This may predispose to cerebral autoregulatory breakthrough and edema formation during increased blood pressure as seen in eclampsia. (C) 2012 International Society for the Study of Hypertension in Pregnancy. Published by Elsevier B.V. All rights reserved.

OBJECTIVE Although patients with type 2 diabetes (T2D) with nephropathy are at high risk for renal and cardiovascular complications, relevant biomarkers have been poorly identified. Because renal impairment may increase biomarker levels, this potentially confounds associations between biomarker levels and risk. To investigate the predictive value of a biomarker in such a setting, we examined baseline levels of growth differentiation factor-15 (GDF-15), N-terminal prohormone of B-type natriuretic peptide (NTproBNP), and high-sensitivity troponin T (hs-TnT) in relation to renal and cardiovascular risk in T2D patients with nephropathy. RESEARCH DESIGN AND METHODS Eight hundred sixty-one T2D patients from the sulodexide macroalbuminuria (Sun-MACRO) trial were included in our post hoc analysis. Prospective associations of baseline serum GDF-15, NTproBNP, and hs-TnT with renal and cardiovascular events were determined by Cox multiple regression and C-statistic analysis. Renal base models included albumin-to-creatinine ratio (ACR), serum creatinine, hemoglobin, age, and sex. Cardiovascular base models included diastolic blood pressure, ACR, cholesterol, age, and sex. RESULTS The mean (±SD) estimated glomerular filtration rate was 33 ± 9 mL/min/1.73 m2, and the median serum concentration for GDF-15 was 3,228 pg/mL (interquartile range 2,345–4,310 pg/mL), for NTproBNP was 380 ng/L (155–989 ng/L), and for hs-TnT was 30 ng/L (20–47 ng/L). In multiple regression analysis, GDF-15 (hazard ratio [HR] 1.83, P = 0.04), NTproBNP (HR 2.34, P = 0.004), and hs-TnT (HR 2.09, P = 0.014) were associated with renal events, whereas NTproBNP (HR 3.45, P < 0.001) was associated with cardiovascular events. The C-statistic was improved by adding NTproBNP and hs-TNT to the renal model (0.793 vs. 0.741, P = 0.04). For cardiovascular events, the C-statistic was improved by adding NTproBNP alone (0.722 vs. 0.658, P = 0.018). CONCLUSIONS Biomarkers GDF-15, NTproBNP, and hs-TnT associate independently with renal risk, whereas NTproBNP independently predicts cardiovascular risk.

The sphingosine-1-phosphate (S1P) analog FTY720 exerts pleiotropic effects on the cardiovascular system and causes down-regulation of S1P receptors. Myogenic constriction is an important mechanism regulating resistance vessel function and is known to be modulated by S1P. Here we investigated myogenic constriction and vascular function of mesenteric arteries of rats chronically treated with FTY720. Wistar rats received FTY720 1mg/kg/daily for six weeks. At termination, blood pressure was recorded and small mesenteric arteries collected for vascular studies in a perfusion set up. Myogenic constriction to increased intraluminal pressure was low, but a sub-threshold dose of S1P profoundly augmented myogenic constriction in arteries of both controls and animals chronically treated with FTY720. Interestingly, endothelial denudation blocked the response to S1P in arteries of FTY720-treated animals, but not in control rats. In acute experiments, presence of FTY720 significantly augmented the contractile response to S1P, an effect that was partially abolished after the inhibition of cyclooxygenase (COX-)-derived prostaglandins. FTY720 down regulated S1P1 but not S1P2 in renal resistance arteries and in cultured human endothelial cells. This study therefore demonstrates the endothelium is able to compensate for the complete loss of responsiveness of the smooth muscle layer to S1P after long term FTY720 treatment through a mechanism that most likely involves enhanced production of contractile prostaglandins by the endothelium.

In healthy rats, the physiological variation of baseline endothelial function of intrarenal arteries correlates with the severity of renal damage in response to a subsequent specific renal injury. However, whether such a variation in endothelial function may also condition or predict the variable response to angiotensin-converting enzyme-inhibiting treatment in these individuals has not been addressed before. To study this, 5/6 nephrectomy was performed to induce renal injury and chronic kidney disease in a group of healthy Wistar rats. At the time of nephrectomy, interlobar arteries were obtained from the extirpated right kidney and studied in vitro for endothelium-dependent relaxation to acetylcholine. Six weeks thereafter, treatment with lisinopril was started ( n = 11) and continued for 9 wk. Proteinuria (metabolic cages) and systolic blood pressure (SBP; tail cuff) were evaluated weekly, and these were analyzed in relation to renal endothelial function at baseline. 5/6 Nephrectomy induced an increase in SBP and progressive proteinuria. Treatment with lisinopril reduced SBP and slowed proteinuria, albeit to a variable degree among individuals. The acetylcholine-induced renal artery dilation at baseline negatively correlated with lisinopril-induced reduction of proteinuria ( r2= 0.648, P = 0.003) and with the decrease in SBP ( r2= 0.592, P = 0.006). Our data suggest that angiotensin-converting enzyme-inhibitor attenuates the progression of renal damage the most in those individuals with decreased basal renal endothelial-mediated vasodilation.

Vavrinec P, Henning RH, Goris M, Vavrincova-Yaghi D, Buikema H, van Dokkum RP. Vascular smooth muscle function of renal glomerular and interlobar arteries predicts renal damage in rats. Am J Physiol Renal Physiol 303: F1187-F1195, 2012. First published July 11, 2012; doi:10.1152/ajprenal.00653.2011.-Previously, it was shown that individuals with good baseline (a priori) endothelial function in isolated (in vitro) renal arteries developed less renal damage after 5/6 nephrectomy (5/6Nx; Gschwend S, Buikema H, Navis G, Henning RH, de Zeeuw D, van Dokkum RP. J Am Soc Nephrol 13: 2909-2915, 2002). In this study, we investigated whether preexisting glomerular vascular integrity predicts subsequent renal damage after 5/6Nx, using in vivo intravital microscopy and in vitro myogenic constriction of small renal arteries. Moreover, we aimed to elucidate the role of renal ANG II type 1 receptor (AT1R) expression in this model. Anesthetized rats underwent intravital microscopy to visualize constriction to ANG II of glomerular afferent and efferent arterioles, with continuous measurement of blood pressure, heart rate, and renal blood flow. Thereafter, 5/6Nx was performed, interlobar arteries were isolated from the extirpated kidney, and myogenic constriction was assessed in a perfused vessel setup. Blood pressure and proteinuria were assessed weekly for 12 wk, and focal glomerulosclerosis (FGS) was determined at the end of study. Relative expression AT1R in the kidney cortex obtained at 5/6Nx was determined by PCR. Infusion of ANG II induced significant constriction of both afferent and efferent glomerular arterioles, which strongly positively correlated with proteinuria and FGS at 12 wk after 5/6Nx. Furthermore, in vitro measured myogenic constriction of small renal arteries negatively correlated with proteinuria 12 wk after 5/6Nx. Moreover, in vivo vascular reactivity negatively correlated with in vitro reactivity. Additionally, relative expression of AT1R positively correlated with responses of glomerular arterioles and with markers of renal damage. Both in vivo afferent and efferent responses to ANG II and in vitro myogenic constriction of small renal arteries in the healthy rat predict the severity of renal damage induced by 5/6Nx. This vascular responsiveness is highly dependent on AT1R expression. Intraorgan vascular integrity may provide a useful tool to guide the prevention and treatment of renal end-organ damage.

Background: Chronic renal failure (CRF) is associated with hypertension, proteinuria, loss of myogenic constriction (MC) of mesenteric arteries and increased production of reactive oxygen species (ROS) under experimental conditions. Previous results showed that ACE (angiotensin-converting enzyme activity) inhibitor therapy is effective in slowing down the progression of disease. Therefore, we wanted to study whether the inverse AT1(angiotensin II type 1) receptor agonist, losartan (LOS) was effective in preventing loss of MC in a rat model of CRF and whether acute ROS scavengers could improve MC.Methods: Rats underwent 5/6 nephrectomy (5/6 Nx) and were treated with vehicle or LOS (20 mg/kg/day; 5/6 Nx + LOS) for 12 weeks. Thereafter, the MC of the mesenteric arteries were measured in the presence and/or absence of tempol and catalase. Systolic blood pressure and proteinuria were measured weekly.Results: Systolic blood pressure and proteinuria in the 5/6 Nx + LOS group were significantly lower than in the 5/6 Nx group. Moreover, the MC of 5/6 Nx + LOS arteries was significantly increased compared with the untreated 5/6 Nx group (maximum MC, 32.3 ± 6.9 vs 8.9 ± 3.8% (p < 0.01)). Tempol + catalase significantly increased the MC in the 5/6 Nx group, but not in the 5/6 Nx + LOS group (increase in MC, 59.7 ± 13.0 (p < 0.05) vs. 17.0 ± 15.1%).Conclusion: These results support the roles of the RAAS (renin—angiotensin—aldosterone system) and ROS in the vascular dysfunction of systemic vessels in CRF.

Objective— Endothelial cell senescence is an important contributor to vascular aging and is increased under diabetic conditions. Here we investigated whether the antidiabetic hormone glucagon-like peptide 1 (GLP-1) could prevent oxidative stress–induced cellular senescence in endothelial cells. Methods and Results— In Zucker diabetic fatty rats, a significant 2-fold higher level of vascular senescence was observed compared with control lean rats. Dipeptidyl-peptidase 4 (DPP-4) inhibition significantly increased GLP-1 levels in these animals and reduced senescence almost to lean animal levels. In vitro studies with human umbilical vein endothelial cells showed that GLP-1 had a direct protective effect on oxidative stress (H 2 O 2 )–induced senescence and was able to attenuate oxidative stress–induced DNA damage and cellular senescence. The GLP-1 analogue exendin-4 provided similar results, whereas exendin fragment 9–39, a GLP-1 receptor antagonist, abolished this effect. Intracellular signaling by the phosphoinositide 3-kinase (PI3K)/Akt survival pathway did not appear to be involved. Further analysis revealed that GLP-1 activates the cAMP response element-binding (CREB) transcription factor in a cAMP/protein kinase A (PKA)–dependent manner, and inhibition of the cAMP/PKA pathway abolished the GLP-1 protective effect. Expression analysis revealed that GLP-1 can induce the oxidative defense genes HO-1 and NQO1 . Conclusion— Dipeptidyl-peptidase 4 inhibition protects against vascular senescence in a diabetic rat model. In vitro studies with human umbilical vein endothelial cells showed that reactive oxygen species–induced senescence was attenuated by GLP-1 in a receptor-dependent manner involving downstream PKA signaling and induction of antioxidant genes.

Background and purpose: High level of plasma catecholamines is a risk factor for vascular diseases such as hypertension and atherosclerosis. Catecholamines induce hypertrophy of vascular smooth muscle through α1-adrenoceptors, which in cell culture involves the transactivation of epidermal growth factor receptor (EGFR). We hypothesized that EGFR transactivation was also involved in contractions of rat aorta mediated by α1-adrenoceptors. Experimental approach: Thoracic aorta was isolated from 12–14 week old male Wistar rats. In vitro aortic contractile responses to cumulative doses of phenylephrine were characterized in the absence and presence of the EGFR kinase inhibitors, AG1478 and DAPH, in intact and endothelium-denuded rings. Involvement of signal transduction pathways was investigated by using heparin and inhibitors of Src, matrix metalloproteinase (MMP), extracellular signal-regulated kinase (ERK)1/2 and phosphatidyl inositol 3-kinase (PI3K). Phosphorylation of EGFR and ERK1/2 was measured after short-term phenylephrine or EGF stimulation in aorta segments in the presence of AG1478 and the PI3K inhibitor, wortmannin. Key results: AG1478 and DAPH concentration dependently attenuated phenylephrine-induced contractile responses in intact or endothelium-denuded aortic rings. Inhibition of PI3K (wortmannin and LY294002) but not heparin or inhibitors of Src or MMP, prevented the effect of AG1478 on the responses to phenylephrine. Phenylephrine induced phosphorylation of EGFR, which was partially blocked by AG1478. Phenylephrine also increased phosphorylation of ERK1/2, time-dependently and was blocked by AG1478 and wortmannin. Conclusions and implications: Contractions of rat thoracic aorta mediated by α1-adrenoceptors involved transactivation of EGFR, mediated via a PI3K and ERK1/2 dependent pathway.

Ochodnicky P, Henning RH, Buikema HJ, de Zeeuw D, Provoost AP, van Dokkum RP. Renal vascular dysfunction precedes the development of renal damage in the hypertensive Fawn-Hooded rat. Am J Physiol Renal Physiol 298: F625-F633, 2010. First published December 9, 2009; doi:10.1152/ajprenal.00289.2009.-It is unknown whether generalized vascular dysfunction precedes the development of kidney disease. Therefore, we studied myogenic constriction and endothelium-mediated dilatory responses in two inbred Fawn-Hooded (FH) rat strains, one of which spontaneously develops hypertension, proteinuria, and glomerulosclerosis (FHH), whereas the other (FHL) does not. Small renal, mesenteric resistance arteries and thoracic aorta isolated from FH rats before (7 wk old) and after the development of mild proteinuria (12 wks old) were mounted in perfused and isometric set-ups, respectively. Myogenic response, endothelium-dependent relaxation, and the contribution of endothelium-mediated dilatory compounds were studied using their respective inhibitors. Myogenic reactivity was assessed constructing pressure-diameter curves in the presence and absence of calcium. At the age of 7 wk, renal arteries isolated from kidneys of FHH rats developed significantly lower myogenic tone compared with FHL, most likely because of excessive cyclo-oxygenase 1-mediated production of constrictive prostaglandins. Consequently, young FHH demonstrated reduced maximal myogenic tone (22 +/- 4.8 vs. 10.8 +/- 2.0%, P = 0.03) and the peak myogenic index (-6.9 +/- 4.8 vs. 0.6 +/- 0.8%/mmHg, P = 0.07 for FHL vs. FHH, respectively). Active myogenic curves obtained in mesenteric arteries isolated from 7-wk-old rats did not differ between either strain, demonstrating a similar level of systemic myogenic tone in FHL and FHH rats. Therefore, before any renal end-organ damage is present, myogenic response seems selectively impaired in renal vasculature of FHH rats. Aortic reactivity did not differ between FHL and FHH at the time points studied. The present study shows that vascular dysfunction in both small renal and systemic arteries precedes renal end-organ damage in a spontaneous model of hypertension-associated renal damage. These early vascular changes might be potentially involved in the increased susceptibility of FHH rats to renal injury.

In humans, the insertion/deletion polymorphism in the angiotensin converting enzyme (ACE) gene accounts for half of the variance in plasma ACE activity. The deletion allele is associated with high plasma ACE activity, cardiovascular disease, and renal disease. In rat, a similar association is found between the B and L alleles of a microsatellite marker in the ACE gene. We identified the B/L variation in the Wistar outbred rat and bred two lines homozygous for the two alleles (WU-B and WU-L). ACE activity was measured in serum, heart, kidney, and aorta homogenates. Immunohistochemistry and ACE mRNA expression were performed in heart, kidney, and aortic tissue. Aortic rings were collected and stimulated with AngI, AngII, and AngI with Lisinopril to measure ACE functional activity by vasoconstrictor response. Serum, heart, and kidney ACE activity and kidney mRNA expression were two-fold higher in WU-B. Kidney staining showed a clear difference in tubular ACE expression, with more staining in WU-B. While in aorta ACE activity and mRNA expression was twofold higher in WU-L, functional conversion of AngI was higher in WU-B, indicating either a functional difference in AngI to AngII conversion between the two alleles due to different splicing or the presence of other factors involved in the conversion that are differentially expressed as the result of differences in the ACE alleles. The newly developed WU-B and WU-L lines show tissue-specific differences in ACE expression and activity. This provides an experimental tool to study the pathophysiologic consequences of differences in ACE alleles in renal and cardiovascular disease.

Diuretics, when added to angiotensin-converting enzyme inhibitors (ACE inhibitors) treatment, can augment the response to ACE inhibitors, but may have adverse effects on renal function, which negatively affect prognosis. While in heart failure rats combined therapy initially improved cardiac function and prognosis, this benefit was completely lost at later stages. We now studied renal effects of adding hydrochlorothiazide to ACE inhibitor after myocardial infarction in rats. Rats were randomized to ACE inhibitor quinapril monotherapy or quinapril with add-on hydrochlorothiazide. Survival was monitored for 14 months. Plasma creatinine, measured at 4 months, was increased by 40% in quinapril with add-on hydrochlorothiazide compared to quinapril. Although overall 14-months mortality was similar in quinapril with add-on hydrochlorothiazide and quinapril, stratification based on plasma creatinine showed increased mortality in the tertile with highest plasma creatinine (P=0.03, Log rank). With add-on hydrochlorotiazide, renal morphology displayed severe renal interstitial lesions; tubular dilatation and fibrosis. Interstitial myofibroblast transformation (alpha-smooth muscle actine staining) was increased at 8 and 14 months, and coincided with collagen deposition and interstitial inflammation (macrophage influx). In rats with quinapril monotherapy or untreated rats, renal structure was normal. Thus, adding hydrochlorotiazide to ACE inhibitor detrimentally affected not only renal function, but also renal structure in rats with myocardial infarction. Altered pharmacokinetics, resulting from a vicious circle of reduced renal function and increased circulating drug levels, may provide an explanation for the adverse renal effects and may exert unfavorable effects on long-term prognosis after myocardial infarction. (C) 2008 Elsevier B.V. All rights reserved.

Background: To evaluate the effect of supraphysiological levels of angiotensin II and selective angiotensin II type 1 receptor ( AT1-receptor) blockade on neointimal formation and systemic endothelial function after stent implantation in the rat abdominal aorta.Methods: Male Wistar rats were randomized to one of three groups; control (n = 8), angiotensin II infusion (n = 9, 200 ng/kg/min), or candesartan cilexetil (n = 8, AT1-receptor blocker; rats received 14.4 mg kg(-1) day(-1)). Stents were implanted in the abdominal aorta. Histological analyses were performed at 4 weeks. Endothelial function was determined in isolated thoracic aortic rings.Results: Neointimal area was increased in the angiotensin II treated group versus the control group, 0.88 mm(2) +/- 0.21 versus 0.66 mm(2) +/- 0.16 (P Conclusions: Supraphysiological levels of angiotensin II aggravates neointimal formation in the stented rat abdominal aorta, and in parallel decreases endothelial function. AT1-receptor blockade does not reduce neointimal formation in rats without supraphysiological angiotensin II levels. (c) 2007 Elsevier Ireland Ltd. All rights reserved.

We investigated the efficacy of oral rosuvastatin treatment to reduce in-stent neointima formation, both in the absence and presence of high levels of the proproliferative substance angiotensin II (Ang II).Drawbacks of current drug-eluting stents include inhibition of reendothelialization, induction of abnormal coronary endothelial function, and, most importantly, late in-stent thrombosis. Statin treatment might be a more subtle approach, with known beneficial vascular effects.Wistar rats were allocated to four treatment groups by two consecutive randomization steps: one to allocate rosuvastatin 0.047% (wt/wt) supplemented rat chow, and one to implant an osmotic minipump releasing Ang II (200 ng/kg). Stents were implanted in the abdominal aorta in all groups. After 4 weeks, in-stent neointima formation and vascular function in the thoracic aorta were determined.In the absence of Ang II, rosuvastatin reduced neointima formation by 23% as compared with control (0.66+/-0.06 versus 0.51+/-0.02 mm2; P0.05). The presence of Ang II enhanced neointimal area by 30%. This was inhibited to the same extent by rosuvastatin (0.88+/-0.06 versus 0.67+/-0.03 mm2; P0.05). In parallel, rosuvastatin improved endothelial-dependent vasodilatation, both in the presence and absence of high levels of Ang II.Ang II infusion increases in-stent neointima formation and decreases endothelial function. We now provide evidence that rosuvastatin effectively inhibits in-stent neointima formation and in parallel improves endothelial dilator function, both in the presence and absence of high Ang II levels.

Background: Several animal studies suggested that the angiotensin II type 2 (AT2) receptor subtype mediates vasodilation, yet the results in human arteries are less well described and more inconsistent. Therefore, we evaluated the role of the AT2 receptor stimulation on the vasotonus of human internal mammary arteries. Methods: Internal mammary arteries were obtained from 50 patients undergoing coronary bypass surgery. The expression of angiotensin II type I (AT1) receptor and AT2 receptor mRNA was determined by using real-time polymerase chain reaction. In addition, angiotensin II and CGP42112A concentration-response curves (concentration range: 10(-10) M to 10(-6) M) were constructed in absence or presence of candesartan (10(-5) M) and/or the AT2 receptor-antagonist PD-123319 (10-6 M) and/or the a receptor antagonist phentolamine. Results: Both AT1 and AT2 receptor protein and mRNA were detected, and higher AT2 receptor mRNA expression levels were associated with increased contractile response to angiotensin II. Angiotensin II caused vasoconstriction up to 41.1 +/- 6.5% of the maximal response to phenylephrine, and PD123319 significantly reduced this response (28.6 +/- 9.6%, P

Women with renal disease progress at a slower rate to end stage renal disease than men. As angiotensin II has both hemodynamic and direct renal effects, we hypothesized that the female protection may result from gender differences in responses to angiotensin II. Therefore, we studied gender differences in response to angiotensin II, during acute (human) and chronic (rats) angiotensin II administration. In young healthy men (n = 18) and women (n = 18) we studied the responses of renal hemodynamics ((125)I-iothalamate and (131)I-Hippuran) and blood pressure to graded angiotensin II infusion (0.3, 1.0, and 3.0 ng/kg/min for 1 h). Men had increased responses of diastolic blood pressure (P = 0.01), mean arterial pressure (P = 0.05), and a more pronounced decrease in effective renal plasma flow (P = 0.009) than women. We measured the changes in proteinuria and blood pressure in response to chronic administration (200 ng/kg/min for 3 weeks) of angiotensin II in rats. Male rats had an increased response of proteinuria compared with females (GEE analysis, P = 0.001). Male, but not female, angiotensin II-treated rats had increased numbers of renal interstitial macrophages compared to sham-treated rats (P < 0.001). In conclusion, gender differences are present in the response to acute and chronic infusion of angiotensin II. Difference in angiotensin II sensitivity could play a role in gender differences in progression of renal disease.

Objectives Chronic renal failure (CRF) is associated with altered systemic arterial tone and hypertension. Myogenic constriction and endothelium-derived hyperpolarizing factor (EDHF)-dependent relaxation represent major vasoregulatory mechanisms in small systemic arteries. Elevated myogenic response and impaired EDHF might participate in the development of essential hypertension; however, their role in CRF-related hypertension is unknown. We investigated whether myogenic response and EDHF are altered in subtotally nephrectornized (sNX) rats and whether these changes are modifiable by chronic treatment with angiotensin-converting enzyme (ACE) inhibitor.Methods In a pressure arteriograph, myogenic constriction and EDHF-mediated relaxation were evaluated in small mesenteric arteries isolated from male Wistar rats 15 weeks after either sham operation (n = 7) (SHAM), sNX (n = 12) or sNX followed by 9 weeks of treatment with lisinopril (sNX + LIS, 2.5 mg/kg, n = 13).Results Surprisingly, myogenic response was reduced in hypertensive CRF rats (maximal myogenic tone: 37 +/- 2 and 18 +/- 4%, P Conclusion Major constrictive (myogenic) and dilatory (EDHF) mechanisms of small systemic arteries are impaired in hypertensive CRF rats. These alterations do not seem to participate in the development of hypertension, being rather directly related to the severity of renal impairment. Both systemic vascular changes might be restored by renoprotective treatment with ACE inhibitor.

Sodium depletion with diuretics augments the efficacy of angiotensin-converting enzyme-inhibitor therapy for hypertension and renal dysfunction, and possibly for left ventricular dysfunction after myocardial infarction. Underlying mechanisms may involve altered angiotensin-converting enzyme-inhibitor pharmacokinetics. We hypothesized that the diuretic hydrochlorothiazide causes increased steady-state levels of the angiotensin-converting enzyme-inhibitors lisinopril and zofenopril in rats with myocardial infarction. Rats were subjected to coronary ligation to induce myocardial infarction. After I week, rats were randomized to 50 mg/kg/day hydrochlorothiazide or control treatment for 3 weeks. The last week, rats received lisinopril or zofenopril in equipotentent dosages (3.3 and 10 mg/kg/day, respectively). Rats were sacrificed at T-max after the last dose of angiotensin-converting enzyme-inhibitor, and tissues were collected for analysis of drug concentrations. Lisinopril concentrations in plasma were significantly increased by hydrochlorothiazide, at unchanged tissue concentrations. This increase could be fully explained by decreased renal function, as evidenced by increased plasma creatinine levels (lisinopril + hydrochlorothiazide 82 +/- 5 mu M versus lisinopril 61 +/- 5 mu M, P

Heart failure after myocardial infarction (MI) is associated with endothelial dysfunction. There is conflicting evidence on the exact nature of this endothelial dysfunction and how endothelium-dependent vasodilation is affected by angiotensin-converting enzyme inhibitor (ACE-I) therapy. Furthermore, consequences of acute ACE-I withdrawal are largely unknown. Therefore, we studied the contribution of nitric oxide (NO) and endothelium-derived hyperpolarizing factor (EDHF) to the effects of ACE-I therapy and its withdrawal on endothelial function in MI rats. Rats were subjected to coronary ligation to induce MI and were assigned to quinapril or vehicle from 2 weeks to 8 months post-MI. In parallel, MI rats treated for 14 months with quinapril were subjected to treatment withdrawal for 0, 4, and 6 weeks. Acetylcholine (ACh)-induced relaxation and underlying endothelium-derived mediators were studied in isolated aortic rings. Long-term quinapril (8 months) resulted in markedly improved endothelium-dependent vasodilation in rats with myocardial infarction, which could be attributed to marked improvement in non-NO/prostanoid-mediated relaxation (ie, EDHF). After 14 months of follow-up, maximum vasodilation was still preserved by quinapril. Withdrawal after 14 months of treatment caused significantly impaired ACh-induced EDHF-mediated relaxation within 4 weeks. A marked reduction in EDHF-mediated relaxation caused this impairment. NO-mediated relaxation was unaffected. These findings highlight the importance of EDHF impairment in development of endothelial dysfunction after myocardial infarction and the possibility of improving EDHF-mediated vasodilation with chronic ACE inhibitor therapy. In addition, withdrawal of chronic ACE inhibition after MI should be considered carefully, as profound endothelial dysfunction may develop rapidly.

Recent evidence has demonstrated that intensive lipid-lowering therapy with a high-dose statin provides significant clinical benefit beyond moderate lipid-lowering therapy. However, dose-dependent effects of short-term statin therapy on vascular function have not been demonstrated. We studied endothelial function and vascular responsiveness to angiotensin II in patients who had coronary artery diseased and were randomized to receive low- or high-dose atorvastatin (10 or 80 mg, respectively) or placebo. Internal thoracic artery segments were obtained during coronary bypass surgery and studied in vitro. Endothelium-dependent vasodilation was increased with atorvastatin therapy (p = 0.035) but was significantly increased further in patients who received 80 mg compared with those who received 10 mg of atorvastatin (p = 0.05). Endothelium improvement was accompanied by decreased vascular response to angiotensin II (p = 0.039). These findings suggest a mechanism for the clinical benefit of intensive lipid-lowering treatment in coronary heart disease. (c) 2005 Elsevier Inc. All rights reserved.

Persistent activation of the renin-angiotensin system leads to downregulation of the angiotensin type-1 receptor, and consequently, to a decreased response to exogenous angiotensin II. In the present study, we investigated the association of angiotensin II responsiveness to clinical outcome after coronary artery bypass grafting (CABG). We studied the responsiveness to exogenous angiotensin II in human thoracic artery preparations of 114 CABG patients. Mean duration of follow-up was 7.3±0.1 years, during which 21 patients experienced a cardiovascular event. A diminished response to angiotensin II remained in multivariate Cox regression analysis, after adjustment for sex, age, blood pressure, and number of diseased coronary arteries, the strongest predictor for cardiovascular events (relative risk, 3.37 [95% confidence interval, 1.20 to 9.51]; P =0.022). Furthermore, diminished response to angiotensin II was associated with an increased mean arterial pressure (102.85±1.38 versus 97.40±1.37; P =0.003) and a nonsignificant increase in angiotensin-converting enzyme activity, suggestive for a persistently activated renin-angiotensin system. In conclusion, these results suggest that in patients undergoing CABG, a diminished vascular responsiveness of the thoracic artery to exogenous angiotensin II is related to an increased risk of future cardiovascular events.

Introduction The angiotensin-converting enzyme (ACE) DD-genotype is associated with increased plasma and myocardial ACE-activity. The influence of the ACE insertion/deletion (I/D) polymorphism on the effects of ACE-inhibition on vascular responses has not been previously described. Materials and methods In the randomised, double-blind QUinapril On Vascular ACE and Determinants of Ischemia Study (QUO VADIS), 149 patients undergoing coronary bypass surgery were randomised to receive either the ACE inhibitor, quinapril, or placebo. In 82 patients, we obtained ACE-genotype, and measured vascular responses to angiotensin II (Ang II) in left internal mammary arteries. Results In the placebo group, the mean maximal vasoconstriction to Ang II was significantly lower in patients with the DD-genotype than in those with the ID/II genotype (36.2±5.11% [n=13] vs. 55.6±4.57% [n=25]; p=0.01). In the quinapril group, the mean maximal vasoconstriction to Ang II was similar [n=8] vs. 57.7±4.07% [n=35]; p=0.85). between DD- and ID/II-genotype (59.6±9.19% Conclusions DD-genotype patients showed decreased vascular responses to Ang II but treatment with quinapril completely restored the decreased vascular response in DD-genotype patients to the same level as II/ID-genotype patients, while no effect of quinapril was demonstrated in the II/ID-genotype patients.

Low dietary sodium (LS) increases the effect of angiotensin-converting enzyme (ACE) inhibitor therapy in patients and experimental models, but mechanisms underlying this enhanced efficacy are largely unknown. Because the benefits of ACE inhibition are mediated to a considerable extent by their effect on the vasculature, we studied whether low sodium alters the vascular effects of ACE inhibition. Baseline functional and morphological characteristics, and endothelium-dependent and -independent dilatory responses were studied in isolated perfused small intrarenal and mesenteric arteries obtained from control rats (CON), rats on LS, lisinopril-treated rats (CON-LIS), or rats treated with lisinopril during LS (LS-LIS). We found, first, that LS-LIS compared with CON-LIS enhances blood pressure reduction. Second, interlobar renal arteries had increased lumen diameter and reduced adrenergic contractility in CON-LIS compared with CON, without additional effects of LS. In contrast, mesenteric arteries were not altered in CON-LIS compared with CON, but became triggered for increased myogenic and adrenergic constriction in LS-LIS. Third, LS-LIS decreased acetylcholine (ACh)-induced vasodilation in both mesenteric and renal arteries compared with CON-LIS. During the latter condition, opposite prostaglandins are involved in the endothelial function of the two different vascular beds, i.e., increased involvement of contractile prostaglandins in ACh-induced vasodilatation in renal arteries, versus dilatory prostaglandins in mesenteric arteries. Whether cause or consequence of the enhanced blood pressure response, our data demonstrate a modifying effect of dietary sodium on vascular effects of ACE inhibition. These findings provide a rationale for further studies addressing the mechanism-of-actions of our therapies to find additional strategies to improve therapy response.

(1) Increased vascular resistance in chronic heart failure (CHF) has been attributed to stimulated neurohumoral systems. However, local mechanisms may also importantly contribute to set arterial tone. Our aim, therefore, was to test whether pressure-induced myogenic constriction of resistance arteries in vitro--devoid of acute effects of circulating factors--is increased in CHF and to explore underlying mechanisms. (2) At 12 weeks after coronary ligation-induced myocardial infarction or SHAM-operations in rats, we studied isolated mesenteric arteries for myogenic constriction, determined as the active constriction (% of passive diameter) in response to stepwise increase in intraluminal pressure (20 - 160 mmHg), in the absence and presence of inhibitors of potentially involved modulators of myogenic constriction. (3) We found that myogenic constriction in mesenteric arteries from CHF rats was markedly increased compared to SHAM over the whole pressure range, the difference being most pronounced at 60 mmHg (24+/-2 versus 4+/-3%, respectively, P

Objectives. To evaluate and compare the functional type and the degree of antagonism of the selective angiotensin II type 1 receptor blockers (ARB) losartan, EXP 3174 (the active metabolite of losartan), valsartan and candesartan in human internal mammary arteries.

BackgroundVascular repair with sutures is associated with disruption of the endothelial lining and subsequent thrombus formation on the intraluminal lesions. This experimental study was designed to determine whether the use of non-penetrating clips improved endothelial preservation.MethodsIn ten female pigs, 25-mm arteriotomies were made in both carotid arteries. The arteriotomies were repaired with jugular vein patches. On the left side, the repair was done with 1·4-mm titanium clips, and on the right side with two running 6/0 polypropylene sutures. Next, the aorta was divided and subsequently repaired with 2-mm clips in five of these pigs, and with two running 5/0 polypropylene sutures in the remaining five pigs. Endothelial function was studied at the anastomotic site in the carotid arteries by determination of endothelium-dependent and -independent relaxatory responses. Morphometric examination of the carotid arteries and inspection of the aortic endothelium were performed by means of scanning electron microscopy.ResultsMaximal endothelium-dependent relaxation to adenosine 5′-diphosphate was less in sutured than in clipped carotid arteries (P < 0·05), while there was no difference in maximal endothelium-independent relaxation to sodium nitrite. This result in clipped carotid arteries was not accompanied by less intimal hyperplasia. Screening of the aortic anastomotic line showed better preservation of endothelial architecture after clip anastomosis. Mean cross-clamp time for carotid patch repair was significantly less when using clips than with sutures.ConclusionThe use of non-penetrating clips for vascular anastomoses preserved endothelial function and structural integrity better than running sutures, although the degree of intimal hyperplasia was similar.

Angiotensin II is known to stimulate cardiac hypertrophy and contractility. Most angiotensin II effects are mediated via membrane bound AT1 receptors. However, the role of myocardial AT1 receptors in cardiac hypertrophy and contractility is still rarely defined. To address the hypothesis that increased myocardial AT1 receptor density causes cardiac hypertrophy apart from high blood pressure we developed a transgenic rat model which expresses the human AT1 receptor under the control of the alpha-myosin heavy-chain promoter specifically in the myocardium. Expression was identified and quantified by northern blot analysis and radioligand binding assays, demonstrating overexpression of angiotensin II receptors in the transgenic rats up to 46 times the amount seen in nontransgenic rats. Coupling of the human AT1 receptor to rat G proteins and signal transduction cascade was verified by sensitivity to GTP-gamma-S and increased sensitivity of intracellular Ca2+ [Ca2+]i to angiotensin II in fluo-3 loaded transgenic cardiomyocytes. Transgenic rats exhibited normal cardiac growth and function under baseline conditions. Pronounced hypertrophic growth and contractile responses to angiotensin II, however, were noted in transgenic rats challenged by volume and pressure overload. In summary, we generated a new transgenic rat model that exhibits an upregulated myocardial AT1 receptor density and demonstrates augmented cardiac hypertrophy and contractile response to angiotensin II after volume and pressure overload, but not under baseline conditions.

We evaluated the role of SH-groups in improvement of endothelial dysfunction with ACE-inhibitors in experimental heart failure. To this end, we compared the vasoprotective effect of chronic treatment with zofenopril (plus SH-group) versus lisinopril (no SH-group), or N-acetylcysteine (only SH-group) in myocardial infarcted (MI) heart failure rats. After 11 weeks of treatment, aortas were obtained and studied as ring preparations for endothelium-dependent and -independent dilatation in continuous presence of indomethacin to avoid interference of vasoactive prostanoids, and the selective presence of the NOS-inhibitor L-NMMA to determine NO-contribution. Total dilatation after receptor-dependent stimulation with acetylcholine (ACh) was attenuated (−49%, P

The QUO VADIS (the effects of QUinapril On Vascular Ace and Determinants of ISchemia) study was a randomized, double-blind, placebo-controlled trial designed to evaluate the effects of long-term angiotensin-converting enzyme (ACE) inhibition on angiotensin II formation in human vasculature. Patients (n = 187) scheduled for coronary artery bypass surgery used study medication 27 +/- 1 days before surgery. Segments of internal mammary arteries were exposed to increasing doses (0.1 nM-1 microM) of angiotensin I and II in organ baths. The rate of local angiotensin II formation is a function of the reciprocal of the difference between the pEC50's of the dose response curves to angiotensin I and II (-log/mol) and of the area between the curves (units). Quinapril (40 mg) and captopril (3 x 50 mg) similarly and significantly reduced mean blood pressure compared with placebo (p = 0.04). Difference between pEC50's was 0.90 +/- 0.08 in quinapril patients compared with 0.60 +/- 0.08 for placebo (p = 0.01); the area between curves was 91 +/- 8 for quinapril patients compared with 67 +/- 8 for placebo (p = 0.03). Angiotensin II formation was decreased to a lesser extent with captopril and was not statistically different from placebo (p = 0.3); the difference between pEC50's was 0.83 +/- 0.15; the area between curves was 84 +/- 12. This is the first randomized study to demonstrate that long-term oral treatment with an ACE inhibitor reduces vascular angiotensin II formation in humans.

1. Angiotensin converting enzyme (ACE) is thought to be the main enzyme to convert antiotensin I to the vasoactive angiotensin II. Recently, in the human heart, it was found that the majority of angiotensin II formation was due to another enzyme, identified as human heart chymase. In the human vasculature however, the predominance of either ACE or non-ACE conversion of angiotensin I remains unclear. 2. To study the effects of ACE- and chymase-inhibition on angiotensin II formation in human arteries, segments of internal mammary arteries were obtained from 37 patients who underwent coronary bypass surgery. 3. Organ bath experiments showed that 100 microM captopril inhibited slightly the response to angiotensin I (pD2 from 7.09+/-0.11-6.79+/-0.10, P

Like human chymase, hamster chymase is an ANG II-forming enzyme, but pathophysiological roles of chymase are still unknown. We determined the functional conversion of ANG I and [Pro11, d-Ala12]ANG I, a chymase-selective substrate, to ANG II in the hamster cardiovascular system. ANG I and [Pro11, d-Ala12]ANG I produced similar dose-dependent pressor responses in conscious hamsters. Captopril and CV-11974, an ANG II type 1 (AT1)-receptor antagonist, inhibited the responses to ANG I; in contrast, the pressor responses to [Pro11,d-Ala12]ANG I were suppressed only by CV-11974. In the isolated aorta, captopril suppressed ANG I-induced contraction by 84%; administration of captopril with either chymostatin or aprotinin eliminated the contraction. [Pro11, d-Ala12]ANG I-induced contraction was not affected by captopril but was attenuated by chymostatin (71%) and aprotinin (57%). CV-11974 abolished the responses to both substrates, whereas PD-123319, an AT2-receptor antagonist, had no effect. In homogenates of the aorta and heart, soybean trypsin inhibitor-inhibitable ANG II formation predominated over captopril- or aprotinin-inhibitable ANG II formation. These data suggest that [Pro11,d-Ala12]ANG I and part of ANG I were functionally converted to ANG II by chymase and other serine protease(s) in hamster vessels, inducing AT1-receptor-mediated vasoconstriction. Biochemical data supported a role for chymase in the alternative pathway.

The renin-angiotensin system is critically involved in the pathogenesis of atherosclerosis. Cholesterol increases vascular responsiveness to angiotensin II by upregulation of the angiotensin 11 type 1 receptor. However, the effects of C-reactive protein (CRP) on vascular responsiveness to angiotensin 11 are unknown. We evaluated, in vitro, the vascular responsiveness of the internal thoracic artery to angiotensin 11 in patients who underwent coronary bypass grafting. We observed that increased preoperative CRP and increased cholesterol levels were strongly related to increased vascular responsiveness to angiotensin 11 (p

In chronic heart failure, the role of endothelial dysfunction is not yet well established. As calcium metabolism plays an important role in the endothelium, it might be suggested that calcium channel blockers influence endothelial function. Although calcium channel blockers are generally contraindicated in chronic heart failure, because they are believed to stimulate neurohumoral mechanisms and to exert negative inotropic effects, recently it has been suggested that amlodipine might have a favorable affect on mortality in patients with heart failure. The mechanism of amlodipine that contributes to this beneficial effect is not known. Therefore we investigated whether 10 weeks of amlodipine treatment could influence endothelial function in rats with congestive heart failure induced by myocardial infarction. The main finding of our study was that amlodipine, when administered for 10 weeks to rats after a myocardial infarction had been induced, had no significant effects on in vitro and in vivo hemodynamics or neurohormones. The effect of amlodipine on endothelium-intact, norepinephrine-precontracted aortic rings appears to differ from the placebo treatment with respect to the endothelium-dependent relaxation, whereas no differences are seen in endothelium-independent relaxation. We conclude that our data do not support a beneficial role of amlodipine on endothelial function in chronic heart failure.

Utibapril is an angiotensin-converting enzyme (ACE) inhibitor with a proposed tissue-specific inhibitory profile. This implies that at a certain dose, utibapril should be able to inhibit tissue ACE activity without affecting plasma ACE. Moreover, if tissue ACE activity is rate limiting, functional conversion of angiotensin I should be decreased. Accordingly, we studied the dose-dependent effect of long-term treatment with utibapril on plasma and tissue ACE. Normal Wistar rats were randomly allocated to oral treatment with different doses of utibapril (0, 2, 10, 50, or 250 mu g/kg/day) for 30 days. Tissue inhibition of ACE was assessed biochemically, whereas functional conversion of angiotensin I was determined in the isolated organ. Utibapril significantly inhibited plasma, renal, and vascular ACE but not ventricular ACE activity. Notably, however, only treatment with the highest dose of utibapril resulted in a significant inhibition of plasma ACE, whereas vascular ACE activity was already significantly inhibited after treatment with a lower dose of utibapril. In accordance, utibapril dose-dependently inhibited the contraction of isolated aortic rings to angiotensin I. Furthermore, angiotensin I-induced decreases in coronary flow in the isolated heart were significantly inhibited after treatment with the higher doses of utibapril. These data suggest the preferential inhibition of vascular ACE by utibapril in normal rats. Furthermore, the dose-dependent inhibition of the functional conversion of angiotensin I indicates that the tissue ACE activity may be rate limiting in vascular beds in rats.

To compare hypertensive end-organ damage in two genetic forms of hypertension we assessed cardiovascular function in two rat strains of genetic hypertension: transgenic rats overexpressing the mouse Ren-2 gene [(TGR(mREN2)27]) and blood pressure matched spontaneously hypertensive rats (SHR). Despite similarly elevated blood pressure, systolic dp/dt (mmHg/s) was more impaired in transgenic rats (3099 +/- 446) than in SHR (3571 +/- 272) and normals (4342 +/- 119; P

Objective: We investigated endothelial dysfunction and the role of angiotensin (Ang)-II type I (AT1-R) and type II (AT2-R) receptor in the changes in the Ang-II sensitivity in experimental preeclampsia in the rat.Methods: Aortic rings were isolated from low dose lipopolysaccharide (LPS) infused pregnant rats (experimental preeclampsia; n=9), saline-infused pregnant rats (n=8), and saline (n=8) and LPS (n=8) infused non-pregnant rats. Endothelium-dependent acetylcholine--mediated relaxation was studied in phenylephrine-preconstricted aortic rings in the presence of vehicle, N-G-nitro-L-arginine methyl ester and/or indomethacin. To evaluate the role for AT1-R and AT2-R in Ang-II sensitivity, full concentration response curves were obtained for Ang-II in the presence of losartan or PD123319. mRNA expression of the AT1-R and AT2-R, eNOS and iNOS, COX1 and COX2 in aorta were evaluated using real-time RT-PCR.Results: The role of vasodilator prostaglandins in the aorta was increased and the role of endothelium-derived hyperpolarizing factor and response of the AT1-R and AT2-R to Ang-II was decreased in pregnant saline infused rats as compared with non-pregnant rats. These changes were not observed during preeclampsia.Conclusion: Pregnancy induced adaptations in endothelial function, which were not observed in the rat model for preeclampsia. This role of lack of pregnancy induced endothelial adaptation in the pathophysiology of experimental preeclampsia needs further investigation.

We hypothesized that angiotensin-converting enzyme (ACE) insertion/deletion polymorphism may be related to arterial phenotypic differences that could explain the adverse effects of deletion polymorphism. Accordingly, contractile responses to angiotensin I and II (0.1 nmol.1(-1)-1 micromol.1(-1), endothelium-dependent relaxation to methacholine (0.01-100 micromol.1(-1), and the effect of NG-monomethyl-L-arginine (L-NMMA; 100 micromol.1(-1) on phenylephrine (10 micromol.1(-1) induced contraction, were studied in isolated rings of internal mammary arteries obtained from patients undergoing coronary bypass surgery. The results were analysed according to the ACE genotype of the patient (II, n = 8; ID, n = 11; DD, n = 9) as well as the presence/absence of either allele. The arteries from patients with the D allele (ID/DD) displayed a lower sensitivity to methacholine (P < 0.05 vs II), which suggested that the capacity of the endothelium for nitric oxide release in response to stimulation was also lower. By contrast, the increase in phenylephrine-induced contraction, by pre-incubation with L-NMMA, was more pronounced in the group with the DD allele (31 +/- 5%) than with the ID (11 +/- 11%) and II alleles (1 +/- 11%, P < 0.05 vs DD), which suggested a higher level of basal nitric oxide release. Finally, the differences in the responses to angiotensin I and II, which were used to evaluate the vascular conversion of angiotensin I, indicated that the level of angiotensin I conversion was higher in patients with the D allele (ID/DD, P < 0.05 vs II). The findings of this study indicate that ACE insertion/deletion polymorphism is related to arterial phenotypic differences in endothelial function and angiotensin I conversion.

In this review, hypotheses are discussed with regard to the role of local, tissue renin-angiotensin systems in the progression of cardiovascular dysfunction. After local renin-angiotensin systems had been described as functionally distinct systems, recent experimental studies have suggested an association between hyperactivity of these local renin-angiotensin systems, and cardiovascular dysfunction. Moreover, the existence of these local renin- angiotensin systems has been confirmed in humans, and early data indicate that the human cardiac renin-angiotensin system may be activated in heart disease. Furthermore, polymorphisms in genes coding for the renin-angiotensin system seem associated with hypertension and left ventricular hypertrophy. These observations may be clinically relevant as inhibition of local renin-angiotensin systems may be an important prerequisite to obtain an optimal clinical effect.