Circulating humanin levels are associated with preserved coronary endothelial function.

Humanin is a small endogenous antiapoptotic peptide, originally identified as protective against Alzheimer's disease, but subsequently also found on human endothelium as well as carotid artery plaques. Endothelial dysfunction is a precursor to the development of atherosclerotic plaques, which are characterized by a highly proinflammatory, reactive oxygen species, and apoptotic milieu. Previous animal studies demonstrated that humanin administration may improve endothelial function. Thus the aim of this study was to test the hypothesis that patients with coronary endothelial dysfunction have reduced systemic levels of humanin. Forty patients undergoing coronary angiography and endothelial function testing were included and subsequently divided into two groups based on coronary blood flow (CBF) response to intracoronary acetylcholine (normal ≥ 50% increase from baseline, n = 20 each). Aortic plasma samples were obtained at the time of catheterization for the analysis of humanin levels and traditional biomarkers of atherosclerosis including C-reactive protein, Lp-Pla(2), and homocysteine. Baseline characteristics were similar in both groups. Patients with coronary endothelial dysfunction (change in CBF = -33 ± 25%) had significantly lower humanin levels (1.3 ± 1.1 vs. 2.2 ± 1.5 ng/ml, P = 0.03) compared with those with normal coronary endothelial function (change in CBF = 194 ± 157%). There was a significant and positive correlation between improved CBF and humanin levels (P = 0.0091) not seen with changes in coronary flow reserve (P = 0.76). C-reactive protein, Lp-Pla(2), and homocysteine were not associated with humanin levels. Thus we observed that preserved human coronary endothelial function is uniquely associated with higher systemic humanin levels, introducing a potential diagnostic and/or therapeutic target for patients with coronary endothelial function.

Beneficial effects of polyphenol-rich olive oil in patients with early atherosclerosis.

PURPOSE: Diets rich in plant-derived polyphenols such as olive oil (OO) and/or catechins such as epigallocatechin 3-gallate (EGCG) have been shown to reduce the incidence of cardiovascular diseases, potentially by improving endothelial function, an important surrogate for atherosclerosis. The possible augmentation of endothelial function with the combined efforts of OO and EGCG is intriguing, yet unknown. METHODS: Eighty-two patients with early atherosclerosis (presence of endothelial dysfunction) were enrolled in this double-blind, randomized trial with 52 completing the study. The aim of the study was to compare the effect of a daily intake of 30 ml simple OO, with 30 ml of EGCG-supplemented OO, on endothelial function as well as on inflammation and oxidative stress after a period of 4 months. Endothelial function was assessed noninvasively via peripheral arterial tonometry (Endo-PAT®). RESULTS: After 4 months, when OO and EGCG-supplemented OO groups were combined, OO significantly improved endothelial function (RHI, 1.59 ± 0.25-1.75 ± 0.45; p < 0.05). However, there were no significant differences in results between the two olive oil groups. Interestingly, with OO supplementation there was a significant reduction in inflammatory parameters: sICAM (196 to 183 ng/mL, p = < 0.001); white blood cells (WBCs) (6.0 × 109/L-5.8 × 109/L, p < 0.05); monocytes (0.48 × 109/L to 0.44 × 109/L, p = 0.05); lymphocytes (1.85 × 109/L to 1.6 × 109/L, p = 0.01); and platelets (242-229 × 109/L, p = 0.047). CONCLUSIONS: Improvement in endothelial dysfunction in patients with early atherosclerosis in association with significant reduction in leukocytes may suggest an important role of early cellular inflammatory mediators on endothelial function. The current study supports one potential mechanism for the role of olive oil, independent of EGCG, modestly supplemented to a healthy cardiovascular diet.

Anti-thrombin therapy during warm ischemia and cold preservation prevents chronic kidney graft fibrosis in a DCD model.

Ischemia reperfusion injury (IRI) is pivotal for renal fibrosis development via peritubular capillaries injury. Coagulation represents a key mechanism involved in this process. Melagatran (M), a thrombin inhibitor, was evaluated in an autotransplanted kidney model, using Large White pigs. To mimic deceased after cardiac death donor conditions, kidneys underwent warm ischemia (WI) for 60 min before cold preservation for 24 h in University of Wisconsin solution. Treatment with M before WI and/or in the preservation solution drastically improved survival at 3 months, reduced renal dysfunction related to a critical reduction in interstitial fibrosis, measured by Sirius Red staining. Tissue analysis revealed reduced expression of transforming growth factor-beta (TGF-beta) and activation level of its effectors phospho-Smad3, Smad4 and connective tissue growth factor (CTGF) after M treatment. Fibrinolysis activation was also observed, evidenced by downregulation of PAI-1 protein and gene expression. In addition, M reduced S100A4 expression and vimentin staining, which are markers for epithelial mesenchymal transition, a major pathway to chronic kidney fibrosis. Finally, expression of oxidative stress markers Nox2 and iNOS was reduced. We conclude that inhibition of thrombin is an effective therapy against IRI that reduces chronic graft fibrosis, with a significantly positive effect on survival.

Urinary mitochondrial DNA copy number identifies renal mitochondrial injury in renovascular hypertensive patients undergoing renal revascularization: A Pilot Study.

AIMS: Patients with renovascular hypertension (RVH) exhibit elevated urinary mtDNA copy numbers, considered to constitute surrogate markers of renal mitochondrial injury. The modest success of percutaneous transluminal renal angioplasty (PTRA) in restoring renal function in RVH has been postulated to be partly attributable to acute reperfusion injury. We hypothesized that mitoprotection during revascularization would ameliorate PTRA-induced renal mitochondrial injury, reflected in elevated urinary mtDNA copy numbers and improve blood pressure and functional outcomes 3 months later. METHODS: We prospectively measured urinary copy number of the mtDNA genes COX3 and ND1 using qPCR in RVH patients before and 24 hrs after PTRA, performed during IV infusion of vehicle (n = 8) or the mitoprotective drug elamipretide (ELAM, 0.05 mg/kg/h, n = 6). Five healthy volunteers (HV) served as controls. Urinary mtDNA levels were also assessed in RVH and normal pigs (n = 7 each), in which renal mitochondrial structure and density were studied ex-vivo. RESULTS: Baseline urinary mtDNA levels were elevated in all RVH patients vs HV and directly correlated with serum creatinine levels. An increase in urinary mtDNA 24 hours after PTRA was blunted in PTRA+ELAM vs PTRA+Placebo. Furthermore, 3-months after PTRA, systolic blood pressure decreased and estimated glomerular filtration rate increased only in ELAM-treated subjects. In RVH pigs, mitochondrial damage was observed using electron microscopy in tubular cells and elevated urinary mtDNA levels correlated inversely with renal mitochondrial density. CONCLUSIONS: PTRA leads to an acute rise in urinary mtDNA, reflecting renal mitochondrial injury that in turn inhibits renal recovery. Mitoprotection might minimize PTRA-associated mitochondrial injury and improve renal outcomes after revascularization.

Micro-RNAS Regulate Metabolic Syndrome-induced Senescence in Porcine Adipose Tissue-derived Mesenchymal Stem Cells through the P16/MAPK Pathway.

Mesenchymal stem cells (MSCs) constitute an important repair system, but may be impaired by exposure to cardiovascular risk factors. Consequently, adipose tissue-derived MSCs from pigs with the metabolic syndrome (MetS) show decreased vitality. A growing number of microRNAs (miRNAs) are recognized as key modulators of senescence, but their role in regulating senescence in MSC in MetS is unclear. We tested the hypothesis that MetS upregulates in MSC expression of miRNAs that can serve as post-transcriptional regulators of senescence-associated (SA) genes. MSCs were collected from swine abdominal adipose tissue after 16 weeks of Lean or Obese diet ( n = 6 each). Next-generation miRNA sequencing (miRNA-seq) was performed to identify miRNAs up-or down-regulated in MetS-MSCs compared with Lean-MSCs. Functional pathways of SA genes targeted by miRNAs were analyzed using gene ontology. MSC senescence was evaluated by p16 and p21 immunoreactivity, H2AX protein expression, and SA-ß-Galactosidase activity. In addition, gene expression of p16, p21, MAPK3 (ERK1) and MAPK14, and MSC migration were studied after inhibition of SA-miR-27b. Senescence biomarkers were significantly elevated in MetS-MSCs. We found seven upregulated miRNAs, including miR-27b, and three downregulated miRNAs in MetS-MSCs, which regulate 35 SA genes, particularly MAPK signaling. Inhibition of miR-27b in cultured MSCs downregulated p16 and MARP3 genes, and increased MSC migration. MetS modulates MSC expression of SA-miRNAs that may regulate their senescence, and the p16 pathway seems to play an important role in MetS-induced MSC senescence.

Effect of gestational hypercholesterolaemia on omental vasoreactivity, placental enzyme activity and transplacental passage of normal and oxidised fatty acids.

OBJECTIVE: Maternal hypercholesterolaemia during pregnancy increases lipid peroxidation in mothers and fetuses and programs increased susceptibility to atherosclerosis later in life. The objective of this study was to elucidate the role of the placenta in mediating oxidative stress from mother to offspring. DESIGN: Comparison between normo- and hypercholesterolaemic mothers (n = 36 each) and their children. SETTING: Obstetric wards, hospitals of the University of Naples and Regione Campania. POPULATION: Healthy primiparas delivering by caesarean section. METHODS: Biochemical measurements of oxidative stress and serum leptin in cord plasma and placenta, immunochemistry of placenta microvessels, and vasoreactivity studies were performed. MAIN OUTCOME MEASURES: Oxidative status (i.e. lipid composition and content of oxidised fatty acids, activity of pro- and antioxidant enzymes, immunohistochemical presence of oxidation-specific epitopes) in maternal and cord blood and in placental tissue, as well as vascular reactivity in omental arteries. RESULTS: Hypercholesterolaemia during pregnancy was associated with extensive changes in fatty acid composition of both maternal and cord blood lipids, sufficient to alter vasoreactivity of omental vessels. Results also indicated that the placenta is not only subject to substantial oxidative stress, but that it may further increase fetal oxidative stress through changes of pro- and antioxidant enzyme activities. CONCLUSIONS: The placenta plays an important role in both transmitting and enhancing pathogenic effects of gestational hypercholesterolaemia.

Altered myocardial microvascular 3D architecture in experimental hypercholesterolemia.

BACKGROUND: Experimental hypercholesterolemia (HC) impairs intramyocardial microvascular function. However, whether this is associated with alterations in microvascular architecture remained unknown. Using a novel 3D micro-CT scanner, we tested the hypothesis that HC is associated with an alteration in the microvascular architecture. METHODS AND RESULTS: Pigs were euthanized after 12 weeks of either normal (n=6) or 2% HC (n=6) diet. The hearts were excised and the coronary arteries injected with a radiopaque contrast material. Myocardial samples were scanned with micro-CT, and 3D images were reconstructed with 21-microm cubic voxels. The myocardium was tomographically subdivided into subepicardium and subendocardium, and microvessels (<500 microm in diameter) were counted in situ within each region. In the subendocardium of HC pigs, the intramyocardial density of microvessels was significantly higher than in normal animals (1221.4+/-199.7 versus 758.3+/-90.8 vessels/cm(3), P:<0.05) because of an increase in the number of microvessels <200 microm in diameter (1214.4+/-199.7 versus 746. 6+/-101.5 vessels/cm(3), P:<0.05). The subepicardial vascular density was similar in both groups. CONCLUSIONS: -HC has differential effects on the spatial density of the subendocardial microvasculature that may play a role in regulation and/or spatial distribution of myocardial blood flow. This study also demonstrates the feasibility of studying myocardial microvascular architecture with micro-CT in pathophysiological states.

Minimally invasive evaluation of coronary microvascular function by electron beam computed tomography.

BACKGROUND: We previously demonstrated that in vivo electron-beam computed tomography (EBCT)-based indicator-dilution methods provide an estimate of intramyocardial blood volume (BV) and perfusion (F), which relate as BV=aF+b radicalF, where a characterizes the recruitable (exchange) and b the nonrecruitable (conduit) component of the myocardial microcirculation. In the present study, we compared BV and F with intracoronary Doppler ultrasound-based coronary blood flow (CBF) as a method for detecting and quantifying differential responses of these microvascular components to vasoactive drugs in normal (control) and hypercholesterolemic (HC) pigs. METHODS AND RESULTS: BV and F values were obtained from contrast-enhanced EBCT studies in 14 HC and 14 control pigs. BV, F, and CBF values were obtained at baseline (intracoronary infusion of saline) and after 5 minutes each of intracoronary infusion of adenosine (100 microgram. kg(-1). min(-1)) and nitroglycerin (40 microgram/min). BV and CBF reserves in response to adenosine were attenuated in HC pigs compared with controls (90+/-36% versus 127+/-42%, P<0.03, and 485+/-182% versus 688+/-160%, P<0.01, respectively). The relationship between BV and F showed consistently lower recruitable BV in HC versus control pigs. Nonrecruitable BV reserve in response to adenosine was attenuated in HC compared with controls (77+/-20% versus 135+/-28%, P<0.001). Our findings are consistent with HC-induced impairment of intramyocardial resistance vessel function. CONCLUSIONS: EBCT technology allows minimally invasive evaluation of intramyocardial microcirculatory function and permits assessment of microvascular BV distribution in different functional components. This method may be of value in evaluating the coronary microcirculation in pathophysiological states such as hypercholesterolemia.

Hypercholesterolemia impairs myocardial perfusion and permeability: role of oxidative stress and endogenous scavenging activity.

OBJECTIVES: We intended to study the effect of hypercholesterolemia (HC) on myocardial perfusion and permeability response to increased cardiac demand. BACKGROUND: Hypercholesterolemia is associated with increased incidence of cardiac events and characterized by impaired coronary vascular function, possibly mediated partly through increased pro-oxidative conditions in plasma and tissue. However, it is yet unclear whether HC is also associated with impaired myocardial perfusion and vascular permeability responses in vivo. METHODS: For 12 weeks pigs were fed a normal, HC or HC diet supplemented daily with antioxidants (HC + AO, 100 IU/kg vitamin E and 1 g vitamin C). Myocardial perfusion and vascular permeability were measured in vivo using electron beam computed tomography before and after cardiac challenge with intravenous adenosine. Plasma and tissue oxidative status was determined ex vivo. RESULTS: Plasma cholesterol increased in all cholesterol-fed pigs but was associated with increased markers of oxidative stress only in HC pigs. Myocardial perfusion increased in response to adenosine in normal and HC + AO (+37 +/- 13% and +58 +/- 22%, respectively, p < 0.05 vs. baseline) but not in HC, whereas vascular permeability index increased only in HC pigs (+ 92 +/- 25%, p = 0.002). In HC animals, tissue endogenous oxygen radical scavengers and antioxidant vitamins were depleted and LDL oxidizability enhanced, but both were normalized in HC + AO pigs. Myocardial perfusion response was directly, and permeability inversely, associated with plasma and tissue vitamin concentrations. CONCLUSIONS: This study demonstrates that experimental HC is associated with blunted myocardial perfusion and increased vascular permeability responses in vivo to increased cardiac demand, which may be partly mediated by a shift in oxidative status.

Simvastatin preserves coronary endothelial function in hypercholesterolemia in the absence of lipid lowering.

Recent evidence suggests that some benefit from the 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors may occur independent of lipid lowering. We aimed to determine the effect of simvastatin on coronary endothelial function, endothelial NO synthase (eNOS) expression, and oxidative stress in experimental hypercholesterolemia (HC) in the absence of cholesterol lowering. Pigs were randomized to 3 experimental groups: normal diet (N group), high cholesterol diet (HC group), and HC diet with simvastatin (HC+S group) for 12 weeks. Low density lipoprotein cholesterol was similarly increased in the HC and HC+S groups compared with the N group. In vitro analysis of coronary large- and small-vessel endothelium-dependent vasorelaxation was performed. The mean vasorelaxation of epicardial vessels to bradykinin was significantly attenuated in the HC group compared with the N group (32.3+/-1.2% versus 42.9+/-1.6%, respectively; P<0.0001). This attenuation was significantly reversed in the HC+S group (38.7+/-1.5%, P<0.005 versus HC group). The maximal vasorelaxation to substance P was significantly attenuated in the HC group compared with the N group (50.5+/-11.9% versus 79.3+/-5.3%, respectively; P<0.05). This attenuated response was normalized in the HC+S group (74.9+/-4.1%, P<0.05 versus HC group). The maximal arteriolar vasorelaxation to bradykinin was also significantly attenuated in the HC group compared with the N group (71.9+/-4.9% versus 96.8+/-1.34%, respectively; P<0.005). This was reversed in the HC+S group (98.4+/-0.6%, P<0.0001 versus HC group). Western blotting of coronary tissue homogenates for eNOS demonstrated a decrease in protein levels in the HC group compared with the N group, with normalization in the HC+S group. Elevation of plasma F(2)-isoprostanes and thiobarbituric acid-reactive substances, markers of oxidative stress, occurred in the HC compared with the N group. These changes were reversed in the HC+S group. In summary, simvastatin preserves endothelial function in coronary epicardial vessels and arterioles in experimental HC (in the absence of cholesterol lowering) in association with an increase in coronary eNOS levels and a decrease in oxidative stress. These alterations may play a role in the reduction in cardiac events after treatment with 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors.

Coronary vasa vasorum neovascularization precedes epicardial endothelial dysfunction in experimental hypercholesterolemia.

OBJECTIVE: Experimental hypercholesterolemia is associated with vasa vasorum neovascularization, unknown to occur before or after initial lesion formation. Thus, this study was performed to determine the temporal course of neovascularization of coronary vasa vasorum in relation to endothelial dysfunction, a hallmark of early atherosclerosis. METHODS: Female domestic pigs were fed a normal diet (Group 1), a hypercholesterolemic diet for 2 and 4 weeks (Group 2), or a hypercholesterolemic diet for 6 and 12 weeks (Group 3). In vitro analysis of relaxation response to bradykinin served as an index for epicardial endothelial function. Spatial pattern and density of coronary vasa vasorum were assessed by three-dimensional microscopic computed tomography. RESULTS: Relaxation response of coronary arteries to bradykinin was normal in both Group 1 (93+/-6%) and Group 2 (89+/-7%) but impaired in Group 3 (71+/-11%; P<0.05 vs. Group 1 and 2). In contrast, density of coronary vasa vasorum was significantly higher in both Group 2 (4.88+/-2.45 per-mm(2)) and Group 3 (4.50+/-1.37 per-mm(2)) compared to Group 1 (2.97+/-1.37 per-mm(2); P<0.05 vs. Group 2 and 3). CONCLUSION: This study demonstrates that coronary vasa vasorum neovascularization occurs within the first weeks of experimental hypercholesterolemia and prior to the development of endothelial dysfunction of the host vessel, suggesting a role for vasa vasorum neovascularization in the initial stage of atherosclerotic vascular disease.

Enhanced coronary vasoconstriction to oxidative stress product, 8-epi-prostaglandinF2 alpha, in experimental hypercholesterolemia.

OBJECTIVES: The F2-isoprostanes are a family of novel prostaglandin isomers and a stable product of in vivo oxidative stress. 8-epi-prostaglandinF2 alpha, a member of this isoprostane family, is a vasoconstrictor and its local release may contribute to the abnormal vasomotor tone associated with hypercholesterolemia. We therefore aimed to outline the role of 8-epi-prostaglandinF2 alpha as a coronary vasoconstrictor in experimental hypercholesterolemia. METHODS AND RESULTS: Pigs were randomized to two experimental groups (each n = 9): normal (N) and high cholesterol (HC) diet. To determine the vasoconstrictive effects of 8-epi-prostaglandinF2 alpha in vitro, doses from 10(-9) to 10(-5) M were used to constrict coronary epicardial rings. Plasma total and LDL cholesterol levels were significantly higher in the HC group compared with the N group (P < 0.005) as were plasma 8-epi-prostaglandinF2 alpha levels (P < 0.001). 8-epi-prostaglandinF2 alpha immunoreactivity was present in the vessel wall in both groups. Normal vessels with intact endothelium (n = 8 rings) contracted to 8-epi-prostaglandinF2 alpha (maximal contraction 15.5 +/- 8.74%). In the HC group, rings with intact endothelium had a greater contractile response to 8-epi-prostaglandinF2 alpha compared to normals (72.3 +/- 7.9%; n = 8; P < 0.0001). This was reversed by preincubation with NOR-3, a NO donor (maximal contraction 6.7 +/- 1.56%; n = 5; P < 0.0001). Enhanced contraction in normal vessels occurred with endothelial denudation (98.4 +/- 3.56%; n = 6; P < 0.0001) and with preincubation of the endothelium-intact rings with L-NMMA (N-monomethyl-L-arginine), an NO synthase inhibitor (85.5 +/- 10.3%, n = 6, P < 0.001). The enhanced contraction seen with hypercholesterolemia did not occur with other prostanoid vasoconstrictors. CONCLUSION: Experimental hypercholesterolemia leads to a significant increase in 8-epi-prostaglandinF2 alpha levels in addition to enhanced 8-epi-prostaglandinF2 alpha-induced coronary vasoconstriction, in vitro. These findings support a role for the F2-isoprostanes in the regulation of coronary vasomotor tone in pathophysiologic states.

In vivo renal vascular and tubular function in experimental hypercholesterolemia.

Hypercholesterolemia (HC) is often associated with impaired peripheral and coronary vascular responses to endothelium-dependent vasodilators, which are probably due to low bioavailability of nitric oxide. To examine the effect of HC on renal vascular and tubular function, 22 domestic pigs were studied after being fed a 12-week normal (n=11) or HC (n=11) diet. Renal regional perfusion and intratubular contrast media concentration in each nephron segment (representing fluid reabsorption) were quantified in vivo with electron-beam computed tomography before and after a suprarenal infusion of either acetylcholine (6 pigs of each diet) or sodium nitroprusside (SNP; 5 pigs of each diet). An increase in cortical perfusion, observed in normal pigs with acetylcholine (+35+/-6%, P=0. 002) and SNP (+12+/-4%, P=0.005), was blunted in the HC group (+8. 8+/-4.0, P=0.01, and -4.6+/-4.0%, P=0.1, respectively, P=0.003 and P=0.005 compared with normal) as was an increase in medullary perfusion (+58+/-21 in normal versus +24+/-11% in HC, P=0.04). A decrease in the intratubular contrast media concentration in the distal tubule and collecting duct of normal pigs was observed in all tubular segments (and was significantly enhanced in the proximal tubule and Henle's loop) in the HC group, which was associated with increased sodium excretion. The tubular and renal excretory responses to SNP were similar between the groups. In conclusion, early experimental HC in the pig attenuates renal perfusion response to both endothelium-dependent and -independent vasodilators possibly because of decreased bioavailability or decreased vascular responsiveness to nitric oxide. This vascular impairment may play a role in maladjusted renovascular responses and contribute to renal damage in later stages of atherosclerosis.

Oxidation-sensitive transcription factors and molecular mechanisms in the arterial wall.

Adaptation to various forms of cellular stress involves signal transduction into the cytoplasm and subsequently into the cellular nucleus, and ultimately alteration of gene regulation and expression. Increased oxidative stress, which is associated with increased production of reactive oxygen species and other radical species, plays a pivotal role in vascular dysfunction and contributes substantially to the structural and functional changes leading to vascular disease progression. Activation of oxidation-sensitive transcription factors and molecular mechanisms can be triggered in the systemic, tissue, cellular, and molecular environments, thereby affecting a multitude of pathophysiological events involved in the pathogenesis of atherosclerosis and other vascular diseases. Radicals per se also participate in the pathophysiological vascular response to shear stress and injury. Among the oxidation-sensitive transcription factors, important roles have been ascribed to nuclear factor-kappaB, c-Myc, and the peroxisome proliferator-activated receptor family. Regulation of nuclear events has also been recently proposed to involve corepressor and coactivator molecules. Identification of the genes that are involved in these processes has been facilitated by recent development of microarray chip techniques, which allow simultaneous evaluation of differential gene expression. As many of the transcription factors or their interactions are redox-regulated, antioxidant intervention may affect their bioactivity.

Impaired renal vascular endothelial function in vitro in experimental hypercholesterolemia.

Hypercholesterolemia (HC) induces alterations in systemic vascular reactivity, which can manifest as an attenuated endothelium-dependent relaxation, partly consequent to an impairment in nitric oxide (NO) activity. To determine whether experimental HC has a similar effect on renal vascular function, renal artery segments obtained from pigs fed a HC (n=5) or normal (n=5) diet were studied in vitro. Endothelium-dependent relaxation was examined using increasing concentrations of acetylcholine (Ach), calcium ionophore A23187, and Ach following pre-incubation with N(G)-monomethyl-L-arginine or L-arginine (L-ARG). The NO-donor diethylamine (DEA) was used to examine smooth muscle relaxation response and cyclic GMP generation in endothelium-denuded vessels. The expression of endothelial NO synthase (eNOS) in the renal arteries was examined using Western blotting. Endothelium-dependent relaxation to Ach was significantly attenuated in the HC group compared to normal (53.3+/-9.1 vs. 98.8+/-3.7%, P<0.005), but normalized after pre-incubation with L-ARG (82.3+/-13.8%, P=0.21). Receptor-independent endothelium-dependent relaxation to A23187 was also significantly blunted in HC (75.2+/-10.5 vs. 115.5+/-4.2%, P<0. 017). Smooth muscle relaxation and cyclic GMP generation in response to DEA were greater in denuded HC vessels, while relaxation of intact vessels to nitroprusside was unaltered. In the HC vessels eNOS was almost undetectable. In conclusion, experimental HC attenuates in vitro endothelium-dependent relaxation of the porcine renal artery, possibly due to low bioavailability of NO. These vascular alterations in HC could play a role in the pathogenesis of renal disease or hypertension, supporting a role for HC as a risk factor for renovascular disease.

Involvement of oxidation-sensitive mechanisms in the cardiovascular effects of hypercholesterolemia.

Hypercholesterolemia is a common clinical metabolic and/or genetic disorder that promotes functional and structural vascular wall injury. The underlying mechanisms for these deleterious effects involve a local inflammatory response and release of cytokines and growth factors. Consequent activation of oxidation-sensitive mechanisms in the arterial wall, modulation of intracellular signaling pathways, increased oxidation of low-density lipoprotein cholesterol, and quenching of nitric oxide can all impair the functions controlled by the vascular wall and lead to the development of atherosclerosis. This cascade represents a common pathological mechanism activated by various cardiovascular risk factors and may partly underlie synergism among them as well as the early pathogenesis of atherosclerosis. Antioxidant intervention and restoration of the bioavailability of nitric oxide have been shown to mitigate functional and structural arterial alterations and improve cardiovascular outcomes. Elucidation of the precise nature and role of early transductional signaling pathways and transcriptional events activated in hypercholesterolemia in children and adults, including mothers during pregnancy, and understanding their downstream effects responsible for atherogenesis may help in directing preventive and interventional measures against atherogenesis and vascular dysfunction.

Cardiac production of angiotensin II and its pharmacologic inhibition: effects on the coronary circulation.

Angiotensin II (AII), produced systemically as well as locally in the heart, affects the coronary circulation, as do consequences of its pharmacologic inhibition. AII is a powerful vasoconstrictor directly acting on vascular smooth muscle cells, modulating sympathetic innervation and calcium ion influx, and releasing other vasoconstrictor factors. In addition to these immediate actions, AII has longer-term biologic actions that influence cardiac endothelial function, vascular smooth muscle cell phenotype expression, and fibroblast proliferation. Moreover, the production of AII is interrelated with the vasodilator substances bradykinin, nitric oxide, and prostaglandins E2 and I2 (prostacyclin). Circulating hormonal actions of AII include fluid retention, direct vasoconstriction, and sympathetic neuromodulation, all resulting in increased left ventricular preload and afterload. Because of these local and hormonal characteristics, AII can immediately affect the myocardial balance of metabolic demand and supply and long term can induce structural vascular and myocardial alterations. Pharmacologic inhibition of AII production likely conveys myocardial and vascular protection in situations of acute myocardial oxygen debt. In the long term, inhibition of AII may attenuate structural changes in the coronary microcirculation related to various cardiomyopathies or acute tissue injury, and direct antiatherogenic effects may also occur.

Novel noninvasive techniques for studying renal function in man.

Renal artery stenosis is a major cause of renovascular hypertension in humans, and may lead to ischemic nephropathy and end-stage renal disease. The mechanisms responsible for the progressive renal functional and structural alterations have not been fully elucidated, partly because of the lack of reliable, noninvasive techniques capable of quantifying renal regional hemodynamics and function distal to a stenosis in the renal artery. Novel imaging tools now enable quantification of concurrent intrarenal (cortical and medullary) hemodynamics, segmental nephron dynamics (intratubular transit times and fluid concentrations), and renal function in the intact kidney. Fast computed tomography (CT) scanners, such as electron beam CT, allow discrimination of subtle alterations in renal perfusion and segmental nephron function consequent to changes in renal perfusion pressure, both within and below the range of renal blood flow autoregulation. This technique provides an opportunity to define intrarenal perfusion patterns and function in animals and patients with renal artery stenosis, and may provide insight into the effects of chronic unilateral or bilateral renovascular disease on both the hypoperfused and contralateral kidneys. This methodology may thereby prove to be very useful in the evaluation of renal disease in general, and the renovascular hypertensive patient in particular.

Quantitation of the in vivo kidney volume with cine computed tomography.

The authors examined the utility of cine computerized tomography (CT) for noninvasive determination of whole kidney, cortical, and medullary volumes. The right kidneys of 14 anesthetized dogs were scanned after an intravenous bolus injection of iohexol, and their volumes determined after boundary identification. After the scans, the kidneys were excised at postmortem examination and their volumes determined by fluid displacement. The mean (+/- standard error of the mean [SEM]) postmortem and in vivo renal volumes were 66.1 +/- 2.2 cc and 78.2 +/- 2.4 cc, respectively (r = 0.86; P less than 0.001). The difference was consistent with the blood, filtrate, and urine contents of the in vivo kidney. The in vivo cortical and medullary volumes correlated poorly with their postmortem volumes because of difficulties in boundary definition. These results demonstrate the feasibility for fast and reliable in vivo whole kidney volume quantitation by cine CT.