Akos Koller

Aging-Induced Phenotypic Changes and Oxidative Stress Impair Coronary Arteriolar Function

We aimed to elucidate the possible role of phenotypic alterations and oxidative stress in age-related endothelial dysfunction of coronary arterioles. Arterioles were isolated from the hearts of young adult (Y, 14 weeks) and aged (A, 80 weeks) male Sprague-Dawley rats. For videomicroscopy, pressure-induced tone of Y and A arterioles and their passive diameter did not differ significantly. In A, arterioles L-NAME (a NO synthase blocker)–sensitive flow-induced dilations were significantly impaired (Y: 41±8% versus A: 3±2%), which could be augmented by superoxide dismutase (SOD) or Tiron (but not l-arginine or the TXA2 receptor antagonist SQ29,548). For lucigenin chemiluminescence, O2·− generation was significantly greater in A than Y vessels and could be inhibited with SOD and diphenyliodonium. NADH-driven O2·− generation was also greater in A vessels. Both endothelial and smooth muscle cells of A vessels produced O2·− (shown with ethidium bromide fluorescence). For Western blotting, expression of eNOS and COX-1 was decreased in A compared with Y arterioles, whereas expressions of COX-2, Cu/Zn-SOD, Mn-SOD, xanthine oxidase, and the NAD(P)H oxidase subunits p47phox, p67phox, Mox-1, and p22phox did not differ. Aged arterioles showed an increased expression of iNOS, confined to the endothelium. Decreased eNOS mRNA and increased iNOS mRNA expression in A vessels was shown by quantitative RT-PCR. In vivo formation of peroxynitrite was evidenced by Western blotting, and immunohistochemistry showing increased 3-nitrotyrosine content in A vessels. Thus, aging induces changes in the phenotype of coronary arterioles that could contribute to the development of oxidative stress, which impairs NO-mediated dilations.

Increased Superoxide Production in Coronary Arteries in Hyperhomocysteinemia Role of Tumor Necrosis Factor-α, NAD(P)H Oxidase, and Inducible Nitric Oxide Synthase

Objective—In coronary arteries, hyperhomocysteinemia (HHcy, a known risk factor for coronary heart disease) impairs flow-induced dilations, which can be reversed by superoxide dismutase (SOD). To evidence increased O2.− generation and elucidate its source, we characterized changes in activity (lucigenin chemiluminescence, hydroethidine staining) and expression of arterial pro- and antioxidant systems (Western blotting, immunohistochemistry, cDNA microarray, reverse-transcription polymerase chain reaction) in the coronary arteries of rats by using methionine diet-induced HHcy. Methods and Results—The increased generation of O2.− by HHcy coronary arteries was inhibited by SOD, diphenyleneiodonium, apocynin, and apocynin plus amino guanidine but was unaffected by allopurinol and rotenone. Also, diphenyleneiodonium-sensitive NADPH-driven O2.− generation was increased in HHcy vessels. In HHcy arteries expression of the smooth muscle-confined NAD(P)H oxidase subunit nox1 and that of iNOS was increased. Expression of p67phox, p22phox, and p47phox subunits and that of endothelial nitric oxide synthase, Cu,Zn-SOD, Mn-SOD, extracellular SOD (mRNA), and xanthine oxidase was unchanged. Microarray analysis showed increased expression of tumor necrosis factor (TNF)-&agr; (confirmed by reverse-transcription polymerase chain reaction, Western blotting, and immunohistochemistry) that was localized in smooth muscle. In vitro incubation (18 hours) of HHcy arteries with anti-TNF-&agr; antibody decreased O2.− production, whereas incubation of control vessels with TNF-&agr; increased O2.− generation and nox1 expression. Conclusions—In coronary arteries, HHcy increases TNF-&agr; expression, which enhances oxidative stress through upregulating a nox1-based NAD(P)H oxidase and inducible nitric oxide synthase. Thus, TNF-&agr; induces a proinflammatory vascular phenotype in HHcy that potentially contributes to the development of coronary atherosclerosis.

Exercise training augments flow-dependent dilation in rat skeletal muscle arterioles : role of endothelial nitric oxide and prostaglandins

We aimed to test the hypothesis that as a consequence of short-term daily exercise, flow (shear stress)-dependent dilation and its mediation by the endothelium are altered in skeletal muscle arterioles. After initial familiarization with the protocol, rats ran on a treadmill once a day (with gradually increasing intensity up to 40 minutes and 28 m/min) for approximately 3 weeks (EX group); a control group remained sedentary (SED group). The active (internal) diameters of isolated gracilis muscle arterioles of SED and EX rats at 80 mm Hg were significantly different (55.2 +/- 2.1 and 49.3 +/- 2.0 microns, P < .05), and their passive diameters (in Ca(2+)-free solution) were 105.3 +/- 3.1 and 111.2 +/- 2.4 microns (not significantly different), respectively. Increases in flow of the perfusion solution from 0 to 12 microL/min elicited a significantly greater increase in diameter of EX arterioles (by 83.5% at maximum flow). This enhanced sensitivity maintained a lower shear stress in EX arterioles (15 to 20 dyne/cm2) compared with SED arterioles (25 to 35 dyne/cm2). In both SED and EX arterioles, flow-dependent dilation was eliminated after removal of the endothelium. Either N omega-nitro-L-arginine, a nitric oxide synthase inhibitor, or indomethacin, an inhibitor of prostaglandin synthesis, shifted the flow-diameter and calculated wall shear stress-diameter curves significantly to the right. Each of the inhibitors reduced flow-dependent dilation to a similar degree (approximately 40% to 45%); their combined administration nearly completely eliminated the dilation of arterioles of both SED and EX rats.(ABSTRACT TRUNCATED AT 250 WORDS)

Aging-induced proinflammatory shift in cytokine expression profile in coronary arteries

The phenotypic and functional changes of coronary arteries with aging promote ischemic heart disease. We hypothesized that these alterations reflect an aging‐induced proinflammatory shift in vascular regulatory mechanisms. Thus, in isolated coronary arteries of young (3‐month‐old) and aged (25‐month‐old) male Fischer 344 rats the expression of 96 cytokines, chemokines, and their receptors were screened by a cDNA‐based microarray technique. In aged vessels expressions of tumor necrosis factor (TNF)‐α (3.3x), interleukin (IL)‐1β (3.0x), IL‐6 (2.9x), IL‐6Rα (2.8x) and IL‐17 (6.1x) genes were significantly increased over young vessels. Quantitative reverse transcriptase‐polymerase chain reaction confirmed these results. Western blotting demonstrated that protein expressions of TNF‐α, IL‐1β, IL‐6, and IL‐17 were also significantly increased in vessels of aged rats compared with those of young rats. Immunofluorescent double labeling showed that in aged vessels IL‐1β and IL‐6 are predominantly localized in the endothelium, whereas TNF‐α and IL‐17 are localized in smooth muscle. Thus, a proinflammatory shift in the profile of vascular cytokine expression may contribute to the aging‐induced phenotypic changes in coronary arteries, promoting the development of ischemic heart disease in the elderly.

Ischaemic heart disease in women: are there sex differences in pathophysiology and risk factors?Position Paper from the Working Group on Coronary Pathophysiology and Microcirculation of the European Society of Cardiology

Cardiovascular disease (CVD) is the leading cause of death in women, and knowledge of the clinical consequences of atherosclerosis and CVD in women has grown tremendously over the past 20 years. Research efforts have increased and many reports on various aspects of ischaemic heart disease (IHD) in women have been published highlighting sex differences in pathophysiology, presentation, and treatment of IHD. Data, however, remain limited. A description of the state of the science, with recognition of the shortcomings of current data, is necessary to guide future research and move the field forward. In this report, we identify gaps in existing literature and make recommendations for future research. Women largely share similar cardiovascular risk factors for IHD with men; however, women with suspected or confirmed IHD have less coronary atherosclerosis than men, even though they are older and have more cardiovascular risk factors than men. Coronary endothelial dysfunction and microvascular disease have been proposed as important determinants in the aetiology and prognosis of IHD in women, but research is limited on whether sex differences in these mechanisms truly exist. Differences in the epidemiology of IHD between women and men remain largely unexplained, as we are still unable to explain why women are protected towards IHD until older age compared with men. Eventually, a better understanding of these processes and mechanisms may improve the prevention and the clinical management of IHD in women.

Regulation of Bone Morphogenetic Protein-2 Expression in Endothelial Cells. Role of Nuclear Factor-κB Activation by Tumor Necrosis Factor-α, H2O2, and High Intravascular Pressure

Background—Recent studies suggest that bone morphogenetic protein-2 (BMP-2), a transforming growth factor-&bgr; superfamily member cytokine, plays an important role both in vascular development and pathophysiological processes, including endothelial activation that is likely to contribute to the development of coronary atherosclerosis, yet the factors that regulate arterial expression of BMP-2 are completely unknown. We tested the hypothesis that BMP-2 expression in endothelial cells is governed by an H2O2 and nuclear factor (NF)-&kgr;&Bgr;–dependent pathway that can be activated by both proinflammatory and mechanical stimuli. Methods and Results—The proinflammatory cytokine tumor necrosis factor (TNF)-&agr; induced NF-&kgr;&Bgr; activation and elicited significant increases in BMP-2 mRNA and protein in primary coronary arterial endothelial cells and human umbilical vein endothelial cells that were prevented by NF-&kgr;&Bgr; inhibitors (pyrrolidine dithiocarbamate and SN-50), silencing of p65 (siRNA), or catalase. Administration of H2O2 also elicited NF-&kgr;&Bgr; activation and BMP-2 induction. In organ culture, exposure of rat arteries to high pressure (160 mm Hg) elicited H2O2 production, nuclear translocation of NF-&kgr;&Bgr;, and upregulation of BMP-2 expression. Although high pressure upregulated TNF-&agr;, it appears that it directly regulates BMP-2 expression, because upregulation of BMP-2 was also observed in vessels of TNF-&agr; knockout mice. Conclusions—Vascular BMP-2 expression can be regulated by H2O2-mediated activation of NF-&kgr;&Bgr; both by inflammatory stimuli and by high intravascular pressure.

Prostaglandins mediate arteriolar dilation to increased blood flow velocity in skeletal muscle microcirculation.

In cremaster muscle of pentobarbital-anesthetized rats, temporary occlusion of an arteriole increased red blood cell velocity (mean increase, 8.2 +/- 1.0 mm/sec from a control velocity of 7.9 +/- 0.7 mm/sec) in proximal parallel arteriolar branches (mean control diameter, 19.4 +/- 0.6 microns). Increases in flow velocity were consistently followed by proportional delayed (6-15 seconds) increases in arteriolar diameter (5.8 +/- 0.7 microns). Administration of NG-monomethyl-L-arginine (200 microM), an inhibitor of the synthesis of endothelium-derived relaxing factor that blocked the arteriolar responses to acetylcholine (1 microM) but not to arachidonic acid (10 microM), did not affect the dilation (mean increase, 8.9 +/- 1.1 microns) due to increases in red blood cell velocity (13.4 +/- 1.5 mm/sec). However, the cyclooxygenase inhibitor indomethacin (or meclofenamate), which completely blocked the dilator response to arachidonic acid but did not change the response to acetylcholine, inhibited the arteriolar dilation (mean increase, 0.3 +/- 0.2 micron) due to increases in red blood cell velocity (9.3 +/- 1.0 mm/sec). Inhibition of prostaglandin synthesis also reduced the increase in calculated blood flow by 57% during occlusion. These results suggest that the arterioles are sensitive to increases in blood flow velocity (wall shear stress), in response to which they release prostaglandins, eliciting vasodilation. The existence of this phenomenon in the skeletal muscle microcirculation suggests a new regulatory mechanism that, by modulation of vascular resistance in the microvascular network, has the role of normalizing wall shear stress and providing for substantial increases in tissue blood flow.

Enhanced Release of Prostaglandins Contributes to Flow-Induced Arteriolar Dilation in eNOS Knockout Mice

Nitric oxide and prostaglandins were shown to contribute to the endothelial mediation of flow-induced dilation of skeletal muscle arterioles of rats. Thus, we hypothesized that flow-induced dilation and its mediation are altered in gracilis muscle arterioles of mice deficient in the gene for endothelial nitric oxide synthase (eNOS-KO) compared with control wild-type (WT) mice. Gracilis muscle arterioles ( approximately 80 micrometer) of male mice were isolated, then cannulated and pressurized in a vessel chamber. The increases in diameter elicited by increases in perfusate flow from 0 to 10 microq/min were similar in arterioles from eNOS-KO (n=28) and WT (n=22) mice ( approximately 20 micrometer at 10 microL/min flow). Removal of the endothelium eliminated flow-induced dilations in vessels of both strains of mice. N(omega)-nitro-L-arginine (L-NNA, 10(-4) mol/L) significantly inhibited flow-induced dilation in arterioles of WT mice by approximately 51% but had no effect on responses of arterioles from eNOS-KO mice. Indomethacin (INDO, 10(-5) mol/L) inhibited flow-induced dilation of WT mice by approximately 49%, whereas it completely abolished this response in arterioles of eNOS-KO mice. Simultaneous administration of INDO and L-NNA eliminated flow-induced responses in arterioles of WT mice. Dilations to carbaprostacyclin were similar at concentrations of 10(-8) and 3x10(-8) mol/L but decreased significantly at 10(-7) mol/L in arterioles of eNOS-KO compared with those of WT mice. These findings demonstrate that, despite the lack of nitric oxide mediation, flow-induced dilation is close to normal in arterioles of eNOS-KO mice because of an enhanced release of endothelial dilator prostaglandins and suggest that this vascular adaptation may contribute to the regulation of peripheral resistance in eNOS-KO mice.

High Pressure Induces Superoxide Production in Isolated Arteries Via Protein Kinase C–Dependent Activation of NAD(P)H Oxidase

Background—Oxidative stress seems to be present in all forms of hypertension. Thus, we tested the hypothesis that high intraluminal pressure (Pi) itself, by activating vascular oxidases, elicits increased superoxide (O2·−) production interfering with flow-induced dilation. Methods and Results—Isolated, cannulated rat femoral arterial branches were exposed in vitro (for 30 minutes) to normal Pi (80 mm Hg) or high Pi (160 mm Hg). High Pi significantly increased vascular O2·− production (as measured by lucigenin chemiluminescence and ethidium bromide fluorescence) and impaired endothelium-dependent dilations to flow; these effects could be reversed by superoxide dismutase. Administration of the NAD(P)H oxidase inhibitor diphenyleneiodonium, apocynin, the protein kinase C (PKC) inhibitor chelerythrine or staurosporin or the removal of extracellular Ca2+ during high Pi treatment prevented the increases in O2·− production, whereas administration of losartan or captopril had no effect. High Pi resulted in significant increases in intracellular Ca2+ ([Ca2+]i) in the vascular wall (fura 2 fluorescence) and phosphorylation of PKC&agr; (Western blotting). The PKC activator phorbol myristate acetate significantly increased vascular O2·− production, which was inhibited by superoxide dismutase, diphenyleneiodonium, chelerythrine, or removal of extracellular Ca2+. Both high Pi and phorbol myristate acetate increased the phosphorylation of the NAD(P)H oxidase subunit p47phox. Conclusion—High Pi itself elicits arterial O2·− production, most likely by PKC-dependent activation of NAD(P)H oxidase, thus providing a potential explanation for the presence of oxidative stress and endothelial dysfunction in various forms of hypertension and the vasculoprotective effect of antihypertensive agents of different mechanisms of action.

Impaired nitric oxide-mediated flow-induced dilation in arterioles of spontaneously hypertensive rats.

We tested the hypothesis that impairment of flow-dependent dilator mechanisms of skeletal muscle arterioles is one of the underlying reasons for the increased peripheral resistance in hypertension. Isolated, cannulated arterioles (approximately 55 microns) of gracilis muscle of 12-week-old spontaneously hypertensive (SH) and normotensive Wistar (NW) rats were investigated. At a constant perfusion pressure (80 mm Hg), the active diameters of NW and SH arterioles were 57.7 +/- 1.9 and 51.5 +/- 3.2 microns, whereas their passive diameters (Ca(2+)-free solution) were 113.6 +/- 2.9 and 101.7 +/- 2.9 microns, respectively. Flow-induced dilation was elicited by increases in flow of the perfusion solution from 0 to 25 microL/min in 5-microL/min steps. This response was significantly less in arterioles of SH compared with NW rats. For example, at 25-microL/min flow, the diameter of arterioles of SH rats was approximately 56% less (P < .05) than those of NW rats. Indomethacin, an inhibitor of prostaglandin synthesis, significantly attenuated the flow-diameter curve in both strains of rats. In contrast, N omega-nitro-L-arginine, a nitric oxide synthase inhibitor, significantly shifted the flow-diameter curve to the right in NW rats, but it did not affect the flow-diameter curve in SH rats. Thus, the present findings demonstrate that in gracilis muscle arterioles of normotensive rats in response to increases in flow (shear stress), prostaglandins and nitric oxide are co-released, resulting in a dilation. In early hypertension, however, there is a reduced arteriolar dilation to increases in flow that is due to the impairment of the nitric oxide-mediated portion of the flow-dependent arteriolar dilation.