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 p67^phox, p22^phox, and p47^phoxsubunits 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)-α (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-α antibody decreased O₂^.−production, whereas incubation of control vessels with TNF-α increased O₂^.−generation and nox1 expression.Conclusions—In coronary arteries, HHcy increases TNF-α expression, which enhances oxidative stress through upregulating a nox1-based NAD(P)H oxidase and inducible nitric oxide synthase. Thus, TNF-α induces a proinflammatory vascular phenotype in HHcy that potentially contributes to the development of coronary atherosclerosis.

—In humans, increased plasma homocysteine (Hcy) has been shown to be correlated with occlusive arterial diseases and atherosclerosis. Studies of isolated conductance vessels of experimental animals suggest that Hcy may interfere with local vasoregulatory mechanisms, yet the effect of hyperhomocysteinemia (HHcy) on the function of microvessels, such as skeletal muscle arterioles, has not been investigated. Male Wistar rats were divided into 2 groups: control rats (C; plasma Hcy, 7.1±0.3 μmol/L; n=25), and rats made HHcy by 1 g/kg body weight daily intake of methionine in the drinking water for 4 weeks (plasma Hcy, 23.6±2.9 μmol/L; P <0.01 versus C; n=25). First-order arterioles (≈130 μm in diameter) were isolated from gracilis muscle, cannulated, and pressurized (80 mm Hg, no-flow conditions). Changes in diameter were observed by videomicroscopy. Arteriolar constrictions to norepinephrine (NE; 3×10 ⁻⁷ mol/L) were significantly ( P <0.01) greater in HHcy compared with C rats (C, 37.7±4.9%; HHcy, 59.5±5.2%). Removal of the endothelium (-E) augmented NE-induced constrictions only in arterioles from C rats, whereas it had no effect on responses of arterioles from HHcy rats (C-E, 55.9±6.9%; HHcy-E, 56.5±7.0%). Dilations to cumulative doses of acetylcholine (ACh; 10 ⁻⁸ mol/L) were significantly reduced in arterioles from HHcy rats (C, 64.0±5.2%; HHcy, 24.1±6.8%). Inhibition of nitric oxide (NO) synthesis with N ^ω -nitro- l -arginine (L-NNA; 10 ⁻⁴ mol/L) significantly decreased ACh-induced dilations of C arterioles, whereas it did not affect HHcy arterioles. Similar alterations were found in arteriolar dilations to histamine, another known NO-dependent agonist. Endothelium-independent dilations to the NO donor sodium nitroprusside were not different in arterioles from C and HHcy rats, either in the presence or absence of L-NNA. Presence of superoxide dismutase and catalase (scavenger of reactive oxygen metabolites) did not affect HHcy-induced alterations in the ACh response. We conclude that hyperhomocysteinemia reduces rat skeletal muscle arteriolar dilations in response to ACh and histamine, and enhances constrictions to NE, alterations that are likely to be caused by the reduced mediation of these responses by NO. The reduced activity of NO in arterioles may contribute to the microvascular impairment described in HHcy.

We hypothesized that changes in hemodynamic forces such as pressure (P) and flow (F) contribute importantly to the development of reactive hyperemia. To exclude the effects of vivo factors, isolated rat skeletal muscle arterioles (∼130 μm) were utilized. We found that changes in P or P + F following occlusions elicited reactive dilations (RD). The peak of RD (up to ∼45 μm), but not the duration of RD, increased to changes in P (80 to 10, then back to 80 mmHg) as a function of the length of occlusions (30, 60, and 120 s). However, changes in P + F (80–10 -80 mmHg + 25–0-25 μl/min) increased both the peak and duration of RD (from ∼25 to 90 s) with longer occlusions. When only P changed, inhibition of nitric oxide synthesis or endothelium removal (E−) reduced only the peak of RD, whereas when P + F were changed, both the peak and duration of RD became reduced. Inhibition of stretch-activated cation channels by gadolinium reduced the peak but enhanced the duration of RD (both to P or P + F) that was unaffected by N ^G-nitro-l-arginine methyl ester (l-NAME) or by E−. When only P changed, inhibition of tyrosine kinases by genistein reduced peak RD but did not affect the RD duration. However, when P + F changed, genistein reduced both the peak and the duration of RD, additional l-NAME reduced the peak RD, but did not affect the duration of RD. Thus in isolated arterioles an RD resembling the characteristics of reactive hyperemia can be generated that is elicited by deformation, stretch, pressure, and flow/shear stress-sensitive mechanisms and is, in part, mediated by nitric oxide.

Elevation of intraluminal pressure increases vasomotor tone, which thought to have a substantial role in regulation of cerebral blood flow (CBF). Interestingly, responses of cerebral vessels to increases in flow varied and have not been studied in human cerebral arteries. We hypothesized that increases in flow elicit constrictions of isolated human and rat cerebral arteries and aimed to elucidate the underlying mechanisms. Human cerebral arteries and rat middle cerebral arteries constricted to increases in flow ( P<0.05). Simultaneous increase in intraluminal flow + pressure further reduced the diameter compared with pressure-induced changes ( P<0.05), leading to constant estimated CBF. Flow-induced constrictions were abolished by HET0016 (inhibitor of synthesis of 20-hydroxyeicosatetraenoic acid (20-HETE) or inhibition of COXs or blocking TP (thromboxane A2/prostaglandin H₂, receptors and attenuated by scavenging reactive oxygen species (ROS). Flow-enhanced ROS formation was significantly reduced by HET0016. In conclusion, in human and rat cerebral arteries (1) increases in flow elicit constrictions, (2) signaling mechanism of flow-induced constriction of cerebral arteries involves enhanced production of ROS, COX activity, and mediated by 20-HETE via TP receptors, and (3) we propose that simultaneous operation of pressure- and flow-induced constrictions is necessary to provide an effective autoregulation of CBF.

Fluid dynamic forces have substantial effects on the movement of lymph and activity of lymph vessels. The effect of increases in intraluminal flow on spontaneous pumping activity of isolated collecting lymphatics has not yet been characterized in a condition in which the intraluminal pressure is constant. Thus, in afferent lymph microvessels isolated from rat iliac lymph nodes, changes in maximum (D_max) and minimum (D_min) diameter to increases in perfusate flow were investigated in the presence of a constant perfusion pressure of 6 cmH₂O. Intraluminal flow was elicited by increases in the difference between outflow and inflow pressures (P_diff, from 0 to 6 cmH₂O). Diameters were measured by videomicroscopy. In response to increases in perfusate flow, D_max and D_min of lymphatics decreased from 157.5 ± 6.1 to 90.9 ± 5.6 μm and from 91.9 ± 5.3 to 66.3 ± 3.6 μm, respectively, whereas vasomotion frequency increased from 18.0 ± 0.7 min⁻¹ to 23.4 ± 1.1 min⁻¹ (at P_diff of 4 cmH₂O). Removal of extracellular Ca²⁺ abolished spontaneous diameter oscillations; under these conditions the passive diameter of lymphatics was 216.0 ± 7.1 μm and did not change in response to increases in perfusion. In the absence of endothelium, flow-induced changes in D_max, D_min, and oscillation frequency were eliminated. N ^ω-nitro-l-arginine methyl ester, an inhibitor of nitric oxide synthase, did not affect flow-induced changes in diameter of lymphatics. In contrast, indomethacin, an inhibitor of prostaglandin synthesis, or SQ-29,548, a PGH₂/thromboxane A₂(PGH₂/TxA₂) receptor blocker, inhibited the perfusion-induced reduction of D_max and D_min of lymphatics and also the increase in the frequency of vasomotion. These findings suggest that the sensitivity of lymphatic endothelium to increases in intraluminal flow could provide an important local intrinsic mechanism for the control of lymphatic resistance by release of constrictor prostanoids PGH₂/TxA₂.

Regular aerobic exercise (RAEX) elicits several positive adaptations in all organs and tissues of the body, culminating in improved health and well-being. Indeed, in over half a century, many studies have shown the benefit of RAEX on cardiovascular outcome in terms of morbidity and mortality. RAEX elicits a wide range of functional and structural adaptations in the heart and its coronary circulation, all of which are to maintain optimal myocardial oxygen and nutritional supply during increased demand. Although there is no evidence suggesting that oxidative metabolism is limited by coronary blood flow (CBF) rate in the normal heart even during maximal exercise, increased CBF and capillary exchange capacities have been reported. Adaptations of coronary macro- and microvessels include outward remodelling of epicardial coronary arteries, increased coronary arteriolar size and density, and increased capillary surface area. In addition, there are adjustments in the neural and endothelial regulation of coronary macrovascular tone. Similarly, there are several adaptations at the level of microcirculation, including enhanced (such as nitric oxide mediated) smooth muscle-dependent pressure-induced myogenic constriction and upregulated endothelium-dependent/shear-stress-induced dilation, increasing the range of diameter change. Alterations in the signalling interaction between coronary vessels and cardiac metabolism have also been described. At the molecular and cellular level, ion channels are key players in the local coronary vascular adaptations to RAEX, with enhanced activation of influx of Ca²⁺ contributing to the increased myogenic tone (via voltage-gated Ca²⁺ channels) as well as the enhanced endothelium-dependent dilation (via TRPV4 channels). Finally, RAEX elicits a number of beneficial effects on several haemorheological variables that may further improve CBF and myocardial oxygen delivery and nutrient exchange in the microcirculation by stabilizing and extending the range and further optimizing the regulation of myocardial blood flow during exercise. These adaptations also act to prevent and/or delay the development of coronary and cardiac diseases.