Hans H. Dietrich

Erythrocytes : Oxygen Sensors and Modulators of Vascular Tone

Through oxygen-dependent release of the vasodilator ATP, the mobile erythrocyte plays a fundamental role in matching microvascular oxygen supply with local tissue oxygen demand. Signal transduction within the erythrocyte and microvessels as well as feedback mechanisms controlling ATP release have been described. Our understanding of the impact of this novel control mechanism will rely on the integration of in vivo experiments and computational models.

Molecular keys to the problems of cerebral vasospasm.

The mechanisms responsible for subarachnoid hemorrhage (SAH)-induced vasospasm are under intense investigation but remain incompletely understood. A consequence of SAH-induced vasospasm, cerebral infarction, produces a nonrecoverable ischemic tissue core surrounded by a potentially amenable penumbra. However, successful treatment has been inconsistent. In this review, we summarize the basic molecular biology of cerebrovascular regulation, describe recent developments in molecular biology to elucidate the mechanisms of SAH-induced vasospasm, and discuss the potential contribution of cerebral microcirculation regulation to the control of ischemia. Our understanding of the pathogenesis of SAH-induced vasospasm remains a major scientific challenge; however, molecular biological techniques are beginning to uncover the intracellular mechanisms involved in vascular regulation and its failure. Recent findings of microvascular regulatory mechanisms and their failure after SAH suggest a role in the development and size of the ischemia. Progress is being made in identifying the various components in the blood that cause SAH-induced vasospasm. Thus, our evolving understanding of the underlying molecular mechanism may provide the basis for improved treatment after SAH-induced vasospasm, especially at the level of the microcirculation.

Cerebrovascular Dysfunction in Amyloid Precursor Protein Transgenic Mice : Contribution of Soluble and Insoluble Amyloid-β Peptide, Partial Restoration via γ-Secretase Inhibition

The contributing effect of cerebrovascular pathology in Alzheimers disease (AD) has become increasingly appreciated. Recent evidence suggests that amyloid-β peptide (Aβ), the same peptide found in neuritic plaques of AD, may play a role via its vasoactive properties. Several studies have examined young Tg2576 mice expressing mutant amyloid precursor protein (APP) and having elevated levels of soluble Aβ but no cerebral amyloid angiopathy (CAA). These studies suggest but do not prove that soluble Aβ can significantly impair the cerebral circulation. Other studies examining older Tg2576 mice having extensive CAA found even greater cerebrovascular dysfunction, suggesting that CAA is likely to further impair vascular function. Herein, we examined vasodilatory responses in young and older Tg2576 mice to further assess the roles of soluble and insoluble Aβ on vessel function. We found that (1) vascular impairment was present in both young and older Tg2576 mice; (2) a strong correlation between CAA severity and vessel reactivity exists; (3) a surprisingly small amount of CAA led to marked reduction or complete loss of vessel function; 4) CAA-induced vasomotor impairment resulted from dysfunction rather than loss or disruption of vascular smooth muscle cells; and 5) acute depletion of Aβ improved vessel function in young and to a lesser degree older Tg2576 mice. These results strongly suggest that both soluble and insoluble Aβ cause cerebrovascular dysfunction, that mechanisms other than Aβ-induced alteration in vessel integrity are responsible, and that anti-Aβ therapy may have beneficial vascular effects in addition to positive effects on parenchymal amyloid.

Mechanism of Extracellular K+-Induced Local and Conducted Responses in Cerebral Penetrating Arterioles

Background and Purpose— Extracellular concentration of potassium ion ([K+]o) may have a significant influence on the cerebral circulation in health and disease. Mechanisms of [K+]o-induced conducted vasomotor responses in cerebral arterioles, possibly linking microvascular regulation to neuronal activity, have not been examined. Methods— We analyzed vascular responses to small increases of [K+]o (up to 5 mmol/L) in isolated, cannulated, and pressurized rat cerebral arterioles (36.5±1.4 &mgr;m). [K+]o was elevated globally through extraluminal application or locally through micropipette, while arteriolar diameter was measured online. Results— Elevation of [K+]o (5 mmol/L) produced dilation that was inhibited by ouabain but not BaCl2. Locally applied [K+]o (3 to 5 mmol/L) produced a biphasic response (initial constriction followed by dilation), both of which were conducted to the remote site (distance 1142±68 &mgr;m). Endothelial impairment inhibited conducted but not local biphasic responses. Extraluminal ouabain attenuated local and conducted secondary dilation but not initial constriction. The local biphasic response was unaffected by extraluminal or intraluminal BaCl2. Extraluminal but not intraluminal BaCl2 impaired both conducted constriction and dilation. Conclusions— In rat penetrating arteriole, (1) [K+]o (3 to 5 mmol/L) strongly regulates arteriolar tone and causes conducted vasomotor responses; (2) local responses to elevated [K+]o are endothelium independent but conducted responses are dependent on an intact endothelium; (3) smooth muscle Na+-K+-ATPase activation is the generator of conducted dilation; and (4) smooth muscle inward rectifier potassium channels sustain conduction. Our findings suggest that potassium-induced conducted vasomotor responses may link local neuronal activity to microvascular regulation, which may be attenuated in pathological conditions.

Contribution of reactive oxygen species to cerebral amyloid angiopathy, vasomotor dysfunction, and microhemorrhage in aged Tg2576 mice

Significance One of the hallmarks of Alzheimer’s disease (AD) is cerebral amyloid angiopathy (CAA), which is a strong and independent risk factor for cerebral hemorrhage, ischemic stroke, and dementia. However, the mechanisms by which CAA contributes to these conditions are poorly understood. Results from the present study provide strong evidence that vascular oxidative stress plays a causal role in CAA-induced cerebrovascular dysfunction, CAA-induced cerebral hemorrhage, and CAA formation, itself. They also suggest that NADPH oxidase is the source of this oxidative stress and that strategies to inhibit NADPH oxidase may have therapeutic potential in patients with AD and CAA. Cerebral amyloid angiopathy (CAA) is characterized by deposition of amyloid β peptide (Aβ) within walls of cerebral arteries and is an important cause of intracerebral hemorrhage, ischemic stroke, and cognitive dysfunction in elderly patients with and without Alzheimer’s Disease (AD). NADPH oxidase-derived oxidative stress plays a key role in soluble Aβ-induced vessel dysfunction, but the mechanisms by which insoluble Aβ in the form of CAA causes cerebrovascular (CV) dysfunction are not clear. Here, we demonstrate evidence that reactive oxygen species (ROS) and, in particular, NADPH oxidase-derived ROS are a key mediator of CAA-induced CV deficits. First, the NADPH oxidase inhibitor, apocynin, and the nonspecific ROS scavenger, tempol, are shown to reduce oxidative stress and improve CV reactivity in aged Tg2576 mice. Second, the observed improvement in CV function is attributed both to a reduction in CAA formation and a decrease in CAA-induced vasomotor impairment. Third, anti-ROS therapy attenuates CAA-related microhemorrhage. A potential mechanism by which ROS contribute to CAA pathogenesis is also identified because apocynin substantially reduces expression levels of ApoE—a factor known to promote CAA formation. In total, these data indicate that ROS are a key contributor to CAA formation, CAA-induced vessel dysfunction, and CAA-related microhemorrhage. Thus, ROS and, in particular, NADPH oxidase-derived ROS are a promising therapeutic target for patients with CAA and AD.

Nitric oxide regulates cerebral arteriolar tone in rats.

Although cerebral penetrating arterioles are main regulators of the brain microcirculation, little is known about the effect of endothelium-derived relaxation factor on these vessels. This study examined the effects of nitric oxide synthase inhibitors on the spontaneous tone of isolated rat cerebral arterioles. Methods Intraparenchymal penetrating arterioles (53 to 102 μm in passive diameter) isolated from Sprague-Dawley rats were cannulated with glass pipettes and subjected to 60 mm Hg of intraluminal pressure. The diameter response to intraluminal and extraluminal treatments was observed with an inverted microscope. Results Extraluminal application of NW-nitro-L-arginine (10−5 mol/L) contracted the arterioles to 63.9±2.8% (P<.05) of the control diameter. This contracting effect was stereospecific and easily reversed by L-arginine dose dependently (10−3, 10−2 mol/L) but not by D-arginine. Intraluminally applied NW-nitro-L-arginine also induced a similar degree of contraction. Another nitric oxide synthase inhibitor, NG-monomethyl L-arginine (10−5, 10−4 mol/L), applied extraluminally induced a dose-dependent contraction to 77.5±6.6% and 68.6±5.4% of the control (P<.05), which was also reversed by L-arginine. L-Arginine alone did not significantly affect vessel diameter, however. Treatment with indomethacin, a cyclooxygenase inhibitor, dilated the vessel to 115.2±7% (P<.05) but did not change the constricting effect of NW-nitro-L-arginine. Conclusions NW-Nitro-L-arginine and NG-monomethyl L-arginine produce substantial contraction in isolated brain arterioles, suggesting that nitric oxide of brain arterioles is continuously produced within the vessel wall. The dilatory effect of indomethacin appears to be independent of the vasoconstriction induced by nitric oxide synthase inhibitor. In these vessels, the effect of nitric oxide synthase inhibitors is not mediated by an indomethacin-sensitive mechanism. A balance probably exists between factors tending to constrict these arterioles and the elaboration of nitric oxide from endothelial cells, which tends to dilate them. The production of nitric oxide from isolated vessels indicates that parenchymal and vessel wall sources of nitric oxide are probably important in brain microcirculatory regulation.

Role of Potassium Channels in Regulation of Brain Arteriolar Tone Comparison of Cerebrum Versus Brain Stem

Background and Purpose— Potassium channels are important regulators of resting tone in large cerebral arteries, but their activity and distribution may vary according to vessel location and species studied. In the cerebral microcirculation in vivo, however, these channels appear to be silent at rest. Our goal was to determine the activity of potassium channels of brain arterioles from 2 origins under basal conditions in vitro. Methods— Penetrating cerebral (40.9±2.2 &mgr;m control diameter) and brain stem (36.2±1.2 &mgr;m) arterioles of rats were prepared from middle cerebral and basilar arteries, respectively. The internal diameter of cannulated and pressurized vessel was monitored with the inverted microscope before and after administration of potassium channel inhibitors. In addition, we studied the effect of nitric oxide synthase inhibition on potassium channel activity. Results— Cerebral and brain stem arterioles were significantly constricted by 4-aminopyridine and low concentration of BaCl2 but not by glibenclamide. The addition of N&ohgr;-nitro-l-arginine to 4-aminopyridine further decreased diameters of both arterioles. Tetraethylammonium ion caused a significant constriction of brain stem but not cerebral arteriole. The brain stem arteriole was further constricted by additional N&ohgr;-nitro-l-arginine. Conclusions— Voltage-dependent and inward-rectifier, but not ATP-sensitive, potassium channels are active under basal conditions of rat cerebral and brain stem arterioles. There is a regional difference in the activity of calcium-activated potassium channels, which, at rest, are open in brain stem but silent in cerebral arterioles. In addition, basal endogenous nitric oxide may not contribute to the activation of voltage-dependent and calcium-activated potassium channels.

Role of Endothelial Nitric Oxide and Smooth Muscle Potassium Channels in Cerebral Arteriolar Dilation in Response to Acidosis

Background and Purpose— Potassium channels or nitric oxide or both are major mediators of acidosis-induced dilation in the cerebral circulation. However, these contributions depend on a variety of factors such as species and vessel location. The present study was designed to clarify whether potassium channels and endothelial nitric oxide are involved in acidosis-induced dilation of isolated rat cerebral arterioles. Methods— Cerebral arterioles were cannulated and monitored with an inverted microscope. Acidosis (pH 6.8 to 7.4) produced by adding hydrogen ions mediated dilation of the cerebral arterioles in a concentration-dependent manner. The role of nitric oxide and potassium channels in response to acidosis was examined with several specific inhibitors and endothelial damage. Results— The dilation was significantly inhibited by potassium chloride (30 mmol/L) and glibenclamide (3 &mgr;mol/L; ATP-sensitive potassium channel inhibitor). We found that 30 &mgr;mol/L BaCl2 (concentration-dependent potassium channel inhibitor) also affected the dilation; however, an additional treatment of 3 &mgr;mol/L glibenclamide did not produce further inhibition. Tetraethylammonium ion (1 mmol/L; calcium-activated potassium channel inhibitor) and 4-aminopyridine (100 &mgr;mol/L; voltage-dependent potassium channel inhibitor) as well as ouabain (10 &mgr;mol/L; Na-K ATPase inhibitor) and N-methylsulphonyl-6-(2-proparglyloxyphenyl) hexanamide (1 &mgr;mol/L; cytochrome P450 epoxygenase inhibitor) did not alter acidotic dilation. N&ohgr;-Monomethyl-l-arginine (10 &mgr;mol/L) and N&ohgr;-nitro-l-arginine (10 &mgr;mol/L) as nitric oxide synthase inhibitor blunted the dilation. Furthermore, the dilation was significantly attenuated after the endothelial impairment. Additional treatment with glibenclamide (3 &mgr;mol/L) further reduced the dilation in response to acidosis. Conclusions— Endothelial nitric oxide and smooth muscle ATP-sensitive potassium channels contribute to acidosis-induced dilation of rat cerebral arterioles. Endothelial damage caused by pathological conditions such as subarachnoid hemorrhage or traumatic brain injury may contribute to reduced blood flow despite injury-induced cerebral acidosis.

G Protein–Coupled Estrogen Receptor Agonist Improves Cerebral Microvascular Function After Hypoxia/Reoxygenation Injury in Male and Female Rats

Background and Purpose— Reduced risk and severity of stroke in adult females are thought to depend on normal levels of endogenous estrogen, which is a known neuro- and vasoprotective agent in experimental cerebral ischemia. Recently, a novel G protein–coupled estrogen receptor (GPER, formerly GPR30) has been identified and may mediate the vasomotor and -protective effects of estrogen. However, the signaling mechanisms associated with GPER in the cerebral microcirculation remain unclear. We investigated the mechanism of GPER-mediated vasoreactivity and also its vasoprotective effect after hypoxia/reoxygenation (H/RO) injury. Methods— Rat cerebral penetrating arterioles from both sexes were isolated, cannulated, and pressurized. Vessel diameters were recorded by computer-aided videomicroscopy. To investigate vasomotor mechanism of the GPER agonist (G-1), several inhibitors with or without endothelial impairment were tested. Ischemia/reperfusion injury was simulated using H/RO. Vasomotor responses to adenosine triphophate after H/RO were measured with or without G-1 and compared with controls. Results— G-1 produced a vasodilatory response, which was partially dependent on endothelium-derived nitric oxide (NO) but not arachidonic acid cascades and endothelial hyperpolarization factor. Attenuation of G-1-vasodilation by the NO synthase inhibitor and endothelium-impairment were greater in vessels from female than male animals. G-1 treatment after H/RO injury fully restored arteriolar dilation to adenosine triphophate compared with controls. Conclusions— GPER agonist elicited dilation, which was partially caused by endothelial NO pathway and induced by direct relaxation of smooth muscle cells. Further, GPER agonist restored vessel function of arterioles after H/RO injury and may play an important role in the ability of estrogen to protect the cerebrovasculature against ischemia/reperfusion injury.

Soluble amyloid-β, effect on cerebral arteriolar regulation and vascular cells

BackgroundEvidence indicates that soluble forms of amyloid-β (Aβ) are vasoactive, which may contribute to cerebrovascular dysfunction noted in patients with Alzheimers Disease and cerebral amyloid angiopathy. The effects of soluble Aβ on penetrating cerebral arterioles - the vessels most responsible for controlling cerebrovascular resistance - have not been studied.ResultsFreshly dissolved Aβ1-40 and Aβ1-42, but not the reverse peptide Aβ40-1 constricted isolated rat penetrating arterioles and diminished dilation to adenosine tri-phosphate (ATP). Aβ1-42 also enhanced ATP-induced vessel constriction. Aβ1-40 diminished arteriolar myogenic response, and an anti-Aβ antibody reduced Aβ1-40 induced arteriolar constriction. Prolonged Aβ exposure in vessels of Tg2576 mice resulted in a marked age-dependent effect on ATP-induced vascular responses. Vessels from 6 month old Tg2576 mice had reduced vascular responses whereas these were absent from 12 month old animals. Aβ1-40 and Aβ1-42 acutely increased production of reactive oxygen species (ROS) in cultured rat cerebro-microvascular cells. The radical scavenger MnTBAP attenuated this Aβ-induced oxidative stress and Aβ1-40-induced constriction in rat arterioles.ConclusionsOur results suggest that soluble Aβ1-40 and Aβ1-42 directly affect the vasomotor regulation of isolated rodent penetrating arterioles, and that ROS partially mediate these effects. Once insoluble Aβ deposits are present, arteriolar reactivity is greatly diminished.