Characterization of a novel model for atherosclerosis imaging: the apolipoprotein E-deficient rat.

BACKGROUND: The apolipoprotein E-deficient (apoE-/-) mouse is a well-established model for studying atherosclerosis. However, its small size limits its use in longitudinal positron emission tomography (PET) imaging studies. Recently, the apoE-/- rat has emerged as an alternative. With this study, we investigate the feasibility of using apoE-/- rats as an in vivo model for longitudinal atherosclerotic PET/CT imaging. RESULTS: ApoE-/- rats showed significantly higher [18F]FDG uptake than controls in the aortic arch (+ 18.5%, p < 0.001) and abdominal aorta (+ 31.0%, p < 0.001) at weeks 12, 26, and 51. ApoE-/- rats exhibited hypercholesterolemia, as evidenced by plasma cholesterol levels that were up to tenfold higher, and total hepatic cholesterol levels that were up to threefold higher than the control rats at the end of the study. Fast protein liquid chromatography cholesterol profiling indicated very high levels of pro-atherogenic apoB-containing very low-density lipoprotein and low-density lipoprotein fractions in the apoE-/- rats. Atherosclerotic lesions cover 19.9% of the surface of the aortic arch (p = 0.0013), and there was a significantly higher subendothelial accumulation of ED1-positive macrophages in the abdominal aorta of the apoE-/- rats compared to control rats (Ctrl) (p = 0.01). No differences in neutral sterols were observed but higher levels of bile acids were found in the apoE-/- rats. CONCLUSION: These data demonstrate early signs of hypercholesterolemia, high levels of bile acids, the development of atherosclerotic lesions, and macrophage accumulation in apoE-/- rats. Therefore, this model shows promise for atherosclerosis imaging studies.

Validation of an ex vivo Flow Model Including Magnetic Resonance Imaging to Study the Effects of Endovascular Treatments on the Arterial Wall.

Endovascular revascularization is the preferred treatment for peripheral arterial disease. Restenosis often occurs as a response to procedure-induced arterial damage. Reducing vascular injury during endovascular revascularization may improve its success rate. This study developed and validated an ex vivo flow model using porcine iliac arteries, obtained from a local abattoir. Twenty arteries (of 10 pigs) were equally allocated to two groups: a mock-treated control group and an endovascular intervention group. Arteries of both groups were perfused with porcine blood for 9 min, including 3 min of balloon angioplasty in the intervention group. Vessel injury was assessed by calculating the presence of endothelial cell denudation, vasomotor function, and histopathological analysis. MR imaging displayed balloon positioning and inflation. Endothelial cell staining showed 76% of denudation after ballooning compared to 6% in the control group (p < 0.001). This was confirmed by histopathological analysis, showing a significantly reduced endothelial nuclei count after ballooning compared to the controls (median: 22 vs. 37 nuclei/mm, p = 0.022). In the intervention group, vasoconstriction and endothelium-dependent relaxation were significantly reduced (p < 0.05).We present an ex vivo flow model to test the effects of endovascular therapy on the vessel's wall morphology, endothelial denudation, and endothelial-dependent vasomotor function under physiological conditions. Additionally, it allows the future testing of human arterial tissue.

Calciprotein Particles Induce Endothelial Dysfunction by Impairing Endothelial Nitric Oxide Metabolism.

BACKGROUND: Calciprotein particles (CPPs) are associated with the development of vascular calcifications in chronic kidney disease. The role of endothelial cells (ECs) in this process is unknown. Here, we investigated the interaction of CPPs and ECs, thereby focusing on endothelial nitric oxide metabolism and oxidative stress. METHODS: CPPs were generated in calcium- and phosphate-enriched medium. Human umbilical vein endothelial cells were exposed to different concentrations of CPPs (0-100 µg/mL) for 24 or 72 hours. Ex vivo porcine coronary artery rings were used to measure endothelial cell-dependent vascular smooth muscle cell relaxation after CPP exposure. Serum samples from an early chronic kidney disease cohort (n=245) were analyzed for calcification propensity (measure for CPP formation) and nitrate and nitrite levels (NOx). RESULTS: CPP exposure for 24 hours reduced eNOS (endothelial nitric oxide synthase) mRNA expression and decreased nitrite production, indicating reduced nitric oxide bioavailability. Also, 24-hour CPP exposure caused increased mitochondria-derived superoxide generation, together with nitrotyrosine protein residue formation. Long-term (72 hours) exposure of human umbilical vein endothelial cells to CPPs induced eNOS uncoupling and decreased eNOS protein expression, indicating further impairment of the nitric oxide pathway. The ex vivo porcine coronary artery model showed a significant reduction in endothelial-dependent vascular smooth muscle cell relaxation after CPP exposure. A negative association was observed between NOx levels and calcification propensity (r=-0.136; P=0.049) in sera of (early) chronic kidney disease patients. CONCLUSIONS: CPPs cause endothelial cell dysfunction by impairing nitric oxide metabolism and generating oxidative stress. Our findings provide new evidence for direct effects of CPPs on ECs and pathways involved.

Endothelial function after the exposition of magnesium degradation products.

One of the most common magnesium (Mg) applications in the biomedical field is in cardiovascular stents. Although Mg is an essential element for homeostasis, Mg is highly reactive, and locally high Mg concentrations can have toxic effects on the surrounding tissue. One strategy to circumvent the Mg toxicity is using coatings or surface modifications that prevent the leaching of excessive Mg ions. In the current study, commercially pure magnesium (c.p Mg) was modified through plasma electrolytic oxidation (PEO) to produce a protective coating primarily composed of Mg oxide (MgO) and Mg hydroxide (Mg(OH)2), which limits leaching of free Mg ions from the base material. As we intend to use this material to produce vascular stents, a biological evaluation of its performance is warranted. Primary human umbilical vein endothelial cells (HUVECs) and smooth muscle cells (SMCs) were the study object. The leaching of free Mg ions from the oxidized materials was investigated, as was its effect on local pH changes. We also investigated the influence of corrosion products, the effects of elevated free Mg concentrations and pH on the cellular behavior on the integrity of monolayers of HUVECs was studied in a static and dynamic model. Results showed that the harmful effect of Mg on cells due to changes in pH and a high concentration of Mg ions could decrease with the influence of flow diffusing corrosion products such as MgO, Mg(OH)2, and H2 among the system. Independently, Mg concentration and pH affected the cell activity of SMCs and HUVECs. Finally, to investigate the influence of leachables on vasomotor function, we exposed porcine aortic rings to PEO-modified Mg stents and assessed endothelial-dependent relaxation. Pure Mg reduced vasorelaxation from 100% in control samples to 30%. Oppositely, PEO-modified Mg did not affect the vasomotor function. Overall, we conclude from this study that the use of PEO coatings reduces the degradation rate of the material reducing the Mg release resulting in better cell viability and vessel function compared to the bare material.

Metformin Improves Endothelial Function and Reduces Blood Pressure in Diabetic Spontaneously Hypertensive Rats Independent from Glycemia Control: Comparison to Vildagliptin.

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.

Predictive Properties of Biomarkers GDF-15, NTproBNP, and hs-TnT for Morbidity and Mortality in Patients With Type 2 Diabetes With Nephropathy.

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.

Parental vitamin D deficiency during pregnancy is associated with increased blood pressure in offspring via Panx1 hypermethylation.

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.

Differential Effects of Long Term FTY720 Treatment on Endothelial versus Smooth Muscle Cell Signaling to S1P in Rat Mesenteric Arteries.

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.

Renal endothelial function is associated with the anti-proteinuric effect of ACE inhibition in 5/6 nephrectomized rats.

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 (r(2) = 0.648, P = 0.003) and with the decrease in SBP (r(2) = 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.

Gender differences in response to acute and chronic angiotensin II infusion: a translational approach.

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.

S1P1 receptor modulation preserves vascular function in mesenteric and coronary arteries after CPB in the rat independent of depletion of lymphocytes.

BACKGROUND: Cardiopulmonary bypass (CPB) may induce systemic inflammation and vascular dysfunction. Sphingosine 1-phosphate (S1P) modulates various vascular and immune responses. Here we explored whether agonists of the S1P receptors, FTY720 and SEW2871 improve vascular reactivity after CPB in the rat. METHODS: Experiments were done in male Wistar rats (total n = 127). Anesthesia was induced by isoflurane (2.5-3%) and maintained by fentanyl and midazolam during CPB. After catheterization of the left femoral artery, carotid artery and the right atrium, normothermic extracorporeal circulation was instituted for 60 minutes. In the first part of the study animals were euthanized after either 1 hour, 1 day, 2 or 5 days of the recovery period. In second part of the study animals were euthanized after 1 day of postoperative period. We evaluated the contractile response to phenylephrine (mesenteric arteries) or to serotonin (coronary artery) and vasodilatory response to acethylcholine (both arteries). RESULTS: Contractile responses to phenylephrine were reduced at 1 day recovery after CPB and Sham as compared to healthy control animals (Emax, mN: 7.9 ± 1.9, 6.5 ± 1.5, and 11.3 ± 1.3, respectively). Mainly FTY720, but not SEW2871, caused lymphopenia in both Sham and CPB groups. In coronary and mesenteric arteries, both FTY720 and SEW2871 normalized serotonin and phenylephrine-mediated vascular reactivity after CPB (p<0.05) and FTY720 increased relaxation to acetylcholine as compared with untreated rats that underwent CPB. CONCLUSION: Pretreatment with FTY720 or SEW2871 preserves vascular function in mesenteric and coronary artery after CPB. Therefore, pharmacological activation of S1P1 receptors may provide a promising therapeutic intervention to prevent CPB-related vascular dysfunction in patients.

Vildagliptin restores renal myogenic function and attenuates renal sclerosis independently of effects on blood glucose or proteinuria in zucker diabetic fatty rat.

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.

Endothelium-dependent relaxation and angiotensin II sensitivity in experimental preeclampsia.

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.

Genetic deletion of growth differentiation factor 15 augments renal damage in both type 1 and type 2 models of diabetes.

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.

Adventitial dissection: a simple and effective way to reduce radial artery spasm in coronary bypass surgery.

OBJECTIVES: 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). CONCLUSIONS: 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.

Renal myogenic constriction protects the kidney from age-related hypertensive renal damage in the Fawn-Hooded rat.

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 (∼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.

Epidermal growth factor receptor inhibitor PKI-166 governs cardiovascular protection without beneficial effects on the kidney in hypertensive 5/6 nephrectomized rats.

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.

Troubleshooting methods for microarray gene expression analysis in the onset of diabetic kidney disease.

INTRODUCTION: Microarrays have become the standard technique for discovering new genes involved in the development of (kidney) disease. Diabetic nephropathy is a frequent complication of diabetes and is characterized by renal fibrosis. As the pathways leading to fibrosis are initiated early in diabetes and in the current study, we aimed at identifying genes associated with renal fibrosis in the first week after induction of diabetes in the rat streptozotocin (STZ) model. METHODS: Conventional microarray analysis methods comparing gene expression to a common reference are not very suitable for time series as gene lists for all time point are very heterogeneous. We therefore sought an analysis technique that would allow us to easily find genes that we either substantially up or down regulated during the first week of diabetes. In the new method, the normalized expression of individual genes was plotted in time. Subsequently, the area under the curve (AUC) was calculated to quantify the overall level of changes in expression of individual genes. RESULTS: AUCs for all genes were plotted in a histogram showing a normal distribution with a mean of close to 0, indicating no change in expression for the majority of genes. Genes with AUCs outside 3 standard deviations of the mean were considered significantly different from control. DISCUSSION: Using this technique, a total of 290 genes were found to be significantly changed in the first week of diabetes. Data on a subset of genes were confirmed by real-time PCR, indicating the validity of the employed new analysis method.

Troubleshooting the rat model of cardiopulmonary bypass: effects of avoiding blood transfusion on long-term survival, inflammation and organ damage.

INTRODUCTION: Rat models of cardiopulmonary bypass (CPB) have been used to examine the mechanisms of associated organ damage and to test intervention strategies. However, these models only partly mimic the clinical situation, because of the use of blood transfusion and arterial inflow via the tail artery. Thus a model using arterial inflow in the aorta and a miniaturized CPB circuit without need of transfusion was validated by examining intra-procedure characteristics, mortality and the effects of CPB on biomarkers of inflammation and cerebral injury during 5days follow-up. METHODS: Male Wistar rats (n=95) were anesthetized with isoflurane (2.5%) and fentanyl/midazolam during CPB. Animals were assigned to Control (n=6), Sham (n=40) or normothermic CPB (n=49) groups. Both Sham and CPB were cannulated in the aorta via the left carotid artery and in the right common jugular vein for access into the right heart. Extracorporeal circulation (ECC) was instituted for 60min only in CPB at a flow rate of 120mLkg(-1)min(-1) employing a CPB circuit of 15ml primed with 6% hydroxyethyl starch 60mgml(-1) solution. Rats were sacrificed at either 1h or 1, 2 or 5days after Sham or weaning from CPB. Plasma IL-6 and s100Beta levels were measured and blood cell counts were performed. RESULTS: Mortality in CPB animals (3 out of 49) and Sham (4 out of 40) did not differ (chi-square=0.46, dF=1, P>0.5). Compared to baseline (1.87±0.46∗10^9cells/L), Sham procedure (cannulation and anesthesia) significantly increased blood neutrophil count at the end of the period matching ECC (6.34±2.36∗10^9cells/L, P<0.05). CPB induced neutrophilia which persisted during 24h recovery. Also, CPB caused a rapid and prominent increase in plasma IL-6 from the first hour of the postoperative period (~1200pg/ml) with continuation (50-90pg/ml) up to 5th day of recovery. S100Beta levels were above detection level only in 3 out of 42 samples from CPB animals. DISCUSSION: Our rat model of CPB without homologous blood transfusion produces a reproducible and reliable systemic inflammatory response, with low mortality rates on long term follow up. The model more closely mimics the human situation in respect to arterial inflow site and avoidance of blood transfusion. Thus, our CPB model is suitable to study its influence on systemic inflammation, ischemia-reperfusion injury, microcirculation and vascular dysfunction in vivo, and to evaluate potential therapeutic interventions.

Structure and function of cerebral and mesenteric resistance arteries in low-dose endotoxin-infused pregnant rats.

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 150mmHg in P-endotoxin (repeated measures ANOVA vs 75mmHg; P<0.05) and between 150 and 175mmHg in P-CTL and NP animals (repeated measures ANOVA vs 75mmHg; P<0.05). PCA myogenic tone was unaffected by pregnancy or endotoxin, however, pregnancy decreased the MA myogenic tone (P<0.05 vs NP). Passive characteristics of PCA and MA were unaffected by pregnancy or endotoxin. 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.