James E. Faber

Vascular contributions to cognitive impairment and dementia including Alzheimer's disease

Scientific evidence continues to demonstrate the linkage of vascular contributions to cognitive impairment and dementia such as Alzheimers disease. In December, 2013, the Alzheimers Association, with scientific input from the National Institute of Neurological Disorders and Stroke and the National Heart, Lung and Blood Institute from the National Institutes of Health, convened scientific experts to discuss the research gaps in our understanding of how vascular factors contribute to Alzheimers disease and related dementia. This manuscript summarizes the meeting and the resultant discussion, including an outline of next steps needed to move this area of research forward.

In situ analysis of alpha-adrenoceptors on arteriolar and venular smooth muscle in rat skeletal muscle microcirculation.

The purpose of this study was to determine whether both α1- and α2-adrenergic receptors exist on vascular smooth muscle of microvessels and whether adrenergic constriction of anatomically distinct microvascular segments is differentially subserved by either receptor subtype. The cremaster skeletal muscle of anesthetized rats was acutely denervated and suspended in a Krebs bath containing cocaine, normetanephrine, and propranolol to block uptake, uptake, and β-receptors, respectively. Intravital microscopy was used to study large distributing arterioles (mean diameter, 100 μ, m), small precapillary arterioles (25 μm), and capacitance venules (140 μm). Concentration-response (diameter change) curves were obtained for bath-added agonists norepinephrine (mixedα1/α2), phenylephrine (α,), and B-HT 933 (α2) in the absence or presence of antagonists prazosin (α1) and yohimbine (α2). Apparent pD2 (-log ED50) values for large arterioles and venules were, respectively, as follows: norepinephrine (7.41 and 7.15), phenylephrine (5.95 and 5.41), and B-HT 933 (5.05 and 5.06). Low concentrations of prazosin (10-8 M) and yohimbine (10-7 M) produced receptor subtype-selective antagonism and parallel, dextral displacement of norepinephrine curves for large arterioles and venules. The large arteriole pK8 (-log KB) was 7.83 ± 0.65 for prazosin and 7.36 ± 0.46 for yohimbine. Higher concentrations of prazosin (10-7 and 3 ± 10-7 M) and yohimbine (10-6 M) produced further dextral but nonparallel displacement of norepinephrine curves. In contrast, receptor subtype-selective concentrations of only yohimbine inhibited adrenergic constriction of small, precapillary arterioles; but prazosin had no effect at receptor subtype-selective concentrations. These data suggest that adrenergic regulation of large arterioles and venules in skeletal muscle uses both α1- and α2-adrenoceptors. Precapillary arterioles, however, may be subserved predominantly by α2-receptors.

Catecholamine-Induced Vascular Wall Growth Is Dependent on Generation of Reactive Oxygen Species

Abstract— &agr;1-Adrenoceptor–dependent proliferation of vascular smooth muscle cells (VSMCs) is strongly augmented by vascular injury, and may contribute to intimal growth and lumen loss. Because reactive oxygen species (ROS) are increased by injury and have been implicated as second messengers in proliferation of VSMCs, we investigated the role of ROS in catecholamine-induced VSMC growth. Rat aortae were isolated 4 days after balloon injury, maintained in organ culture under circumferential wall tension, and exposed to agents for 48 hours. The antioxidants N-acetylcysteine (NAC, 10 mmol/L) and Tiron (5 mmol/L) and the flavin-inhibitor diphenylene iodonium (DPI, 20 &mgr;mol/L) abolished norepinephrine-induced increases in protein synthesis and DNA content in media. In aortic sections, norepinephrine augmented ROS production (dihydroethidium confocal microscopy), which was dose-dependently inhibited by NAC, Tiron, and DPI. In cultured VSMCs, phenylephrine caused time- and dose-dependent ROS generation (aconitase activity), had similar efficacy to thrombin (1 U/mL), and was eliminated by the superoxide dismutase (SOD) mimetic Mn-(III)-tetrakis-(4-benzoic-acid)-porphyrin-chloride (200 &mgr;mol/L) and Tiron. Phenylephrine-induced ROS production and increases in DNA and protein content were blocked by prazosin (0.3 &mgr;mol/L) and abolished in p47phox−/− cells. PEG-SOD (25 U/mL) had little effect, whereas PEG-catalase (50 U/mL) eliminated phenylephrine-induced proliferation in VSMCs. DPI (10 &mgr;mol/L) and apocynin (30 &mgr;mol/L) abolished phenylephrine-stimulated mitogenesis, whereas inhibitors of other intracellular ROS sources had not effect. Furthermore, PE increased p47phox expression (RT-PCR). These data demonstrate that the trophic effect of catecholamines on vascular wall cells is dependent on a ROS-sensitive step that we hypothesize consists of activation of the NAD(P)H-dependent vascular oxidase.

Wide genetic variation in the native pial collateral circulation is a major determinant of variation in severity of stroke

Severity of stroke varies widely among individuals. Whether differences in the extent of the native (preexisting) pial collateral circulation exist and contribute to this variability is unknown. We addressed these questions and probed for potential genetic contributions using morphometric analysis of the collateral circulation in 15 inbred mouse strains recently shown to exhibit wide differences in infarct volume. Morphometrics were determined in the unligated left hemisphere (for native collaterals) and ligated right hemisphere (for remodeled collaterals) 6 days after permanent middle cerebral artery (MCA) occlusion. Variation among strains in native collateral number, diameter, MCA, anterior cerebral artery (ACA), and posterior cerebral artery (PCA) tree territories were, respectively: 56-fold, 3-fold, 42%, 56%, and 61%. Collateral length (P<0.001) and the number of penetrating arterioles branching from them also varied (P<0.05). Infarct volume correlated inversely with collateral number (P<0.0001), diameter (P<0.0001), and penetrating arteriole number (P<0.05) and directly with MCA territory (P<0.05). Relative collateral conductance and MCA territory, when factored together, strongly predicted infarct volume (P<0.0001). Outward remodeling of collaterals in the ligated hemisphere varied ∼3-fold. These data show that the extent of the native pial collateral circulation and collateral remodeling after obstruction vary widely with genetic background, and suggest that this variability, due to natural polymorphisms, is a major contributor to variability in infarct volume.

Vascular Endothelial Growth Factor-A Specifies Formation of Native Collaterals and Regulates Collateral Growth in Ischemia

The density of native (preexisting) collaterals and their capacity to enlarge into large conduit arteries in ischemia (arteriogenesis) are major determinants of the severity of tissue injury in occlusive disease. Mechanisms directing arteriogenesis remain unclear. Moreover, nothing is known about how native collaterals form in healthy tissue. Evidence suggests vascular endothelial growth factor (VEGF), which is important in embryonic vascular patterning and ischemic angiogenesis, may contribute to native collateral formation and arteriogenesis. Therefore, we examined mice heterozygous for VEGF receptor-1 (VEGFR-1+/−), VEGF receptor-2 (VEGFR-2+/−), and overexpressing (VEGFhi/+) and underexpressing VEGF-A (VEGFlo/+). Recovery from hindlimb ischemia was followed for 21 days after femoral artery ligation. All statements below are P<0.05. Compared to wild-type mice, VEGFR-2+/− showed similar: ischemic scores, recovery of hindlimb perfusion, pericollateral leukocytes, collateral enlargement, and angiogenesis. In contrast, VEGFR-1+/− showed impaired: perfusion recovery, pericollateral leukocytes, collateral enlargement, worse ischemic scores, and comparable angiogenesis. Compared to wild-type mice, VEGFlo/+ had 2-fold lower perfusion immediately after ligation (suggesting fewer native collaterals which was confirmed by angiography) and blunted recovery of perfusion. VEGFhi/+ mice had 3-fold greater perfusion immediately after ligation, more native collaterals, and improved recovery of perfusion. These differences were confirmed in the cerebral pial cortical circulation where, compared to VEGFhi/+ mice, VEGFlo/+ formed fewer collaterals during the perinatal period when adult density was established, and had 2-fold larger infarctions after middle cerebral artery ligation. Our findings indicate VEGF and VEGFR-1 are determinants of arteriogenesis. Moreover, we describe the first signaling molecule, VEGF-A, that specifies formation of native collaterals in healthy tissues.

ATP modulates load-inducible IL-1β, COX 2, and MMP-3 gene expression in human tendon cells

Tendon cells receive mechanical signals from the load bearing matrices. The response to mechanical stimulation is crucial for tendon function. However, overloading tendon cells may deteriorate extracellular matrix integrity by activating intrinsic factors such as matrix metalloproteinases (MMPs) that trigger matrix destruction. We hypothesized that mechanical loading might induce interleukin‐1beta (IL‐1β) in tendon cells, which can induce MMPs, and that extracellular ATP might inhibit the load‐inducible gene expression. Human tendon cells isolated from flexor digitorum profundus tendons (FDPs) of four patients were made quiescent and treated with ATP (10 or 100 μM) for 5 min, then stretched equibiaxially (1 Hz, 3.5% elongation) for 2 h followed by an 18‐h‐rest period. Stretching induced IL‐1β, cyclooxygenase 2 (COX 2), and MMP‐3 genes but not MMP‐1. ATP reduced the load‐inducible gene expression but had no effect alone. A medium change caused tendon cells to secrete ATP into the medium, as did exogenous UTP. The data demonstrate that mechanical loading induces ATP release in tendon cells and stimulates expression of IL‐1β, COX 2, and MMP‐3. Load‐induced endogenous IL‐1β may trigger matrix remodeling or a more destructive pathway(s) involving IL‐1β, COX 2, and MMP‐3. Concomitant autocrine and paracrine release of ATP may serve as a negative feedback mechanism to limit activation of such an injurious pathway. Attenuation or failure of this negative feedback mechanism may result in the progression to tendinosis.

Transactivation of Epidermal Growth Factor Receptor Mediates Catecholamine-Induced Growth of Vascular Smooth Muscle

Stimulation of &agr;1-adrenoceptors induces proliferation of vascular smooth muscle cells (SMCs) and contributes to arterial remodeling. Although activation of NAD(P)H oxidase and generation of reactive oxygen species (ROS) are required, little is known about this pathway. In this study, we examined the hypothesis that epidermal growth factor receptor (EGFR) transactivation and extracellular regulated kinases (ERK) are involved in &agr;1-adrenoceptor–mediated SMC growth. Phenylephrine increased protein synthesis in association with a rapid (≤5 minutes) and sustained (≥60 minutes) doubling of phosphorylation of EGFR and ERK1/2, but not p38 or JNK in the media of rat aorta maintained in organ culture. Antagonists of EGFR phosphotyrosine activity (AG-1478) and ERK phosphorylation (PD-98059, U-0126) abolished phenylephrine-induced protein synthesis, whereas antagonists of p38 or JNK phosphorylation had no specific effect. A competitive antagonist (P22) for heparin binding EGF-like growth factor (HB-EGF) blocked phenylephrine-induced protein synthesis, as did downregulation of pro-HB-EGF (CRM197). Phenylephrine-induced protein synthesis was inhibited by neutralizing antibody to HB-EGF and absent in HB-EGF−/− SMCs. Inhibitors of metalloproteinases (BiPS, KB-R7785) also blocked adrenergic growth. The neutralizing antibody against HB-EGF had no effect on the two-fold increase in ROS generation induced by phenylephrine (DCF fluorescence), suggesting that stimulation of NAD(P)H oxidase by &agr;1-adrenoceptor occupation precedes HB-EGF release. Cell culture studies confirmed and extended these findings. These data suggest that &agr;1-adrenoceptor–mediated SMC growth requires ROS-dependent shedding of HB-EGF, transactivation of EGFR, and activation of the MEK1/2-dependent MAP kinase pathway. This trophic pathway may link sympathetic activity to arterial wall growth in adaptive remodeling and hypertrophic disease.

Aging Causes Collateral Rarefaction and Increased Severity of Ischemic Injury in Multiple Tissues

Objective—Aging is a major risk factor for increased ischemic tissue injury. Whether collateral rarefaction and impaired remodeling contribute to this is unknown. We quantified the number and diameter of native collaterals and their remodeling in 3-, 16-, 24-, and 31-month-old mice. Methods and Results—Aging caused an “age-dose-dependent” greater drop in perfusion immediately after femoral artery ligation, followed by a diminished recovery of flow and increase in tissue injury. These effects were associated with a decline in collateral number, diameter, and remodeling. Angiogenesis was also impaired. Mechanistically, these changes were not accompanied by reduced recruitment of T cells or macrophages to remodeling collaterals. However, endothelial nitric oxide synthase signaling was dysfunctional, as indicated by increased protein nitrosylation and less phosphorylated endothelial nitric oxide synthase and vasodilator-stimulated phosphoprotein in collateral wall cells. The cerebral circulation exhibited a similar age-dose-dependent loss of collateral number and diameter and increased tortuosity, resulting in an increase in collateral resistance and infarct volume (eg, 6- and 3-fold, respectively, in 24-month-old mice) after artery occlusion. This was not associated with rarefaction of similarly sized arterioles. Collateral remodeling was also reduced. Conclusion—Our findings demonstrate that aging causes rarefaction and insufficiency of the collateral circulation in multiple tissues, resulting in more severe ischemic tissue injury.

Endothelial Nitric Oxide Synthase Deficiency Causes Collateral Vessel Rarefaction and Impairs Activation of a Cell Cycle Gene Network During Arteriogenesis

Rationale: The collateral circulation is tissue- and life-saving in obstructive arterial disease. Disappointing outcomes in clinical trials aimed at augmenting collateral growth highlight the need for greater understanding of collateral biology. Objective: The role of endothelial nitric oxide synthase (eNOS) in forming native (preexisting) collaterals and remodeling in obstructive disease are unknown or controversial issues, respectively. Methods and Results: We compared the native collateral circulation in healthy tissue and collateral remodeling after femoral artery ligation (FAL) in wild-type and eNOS-knockout (KO) mice. Perfusion after FAL fell further in adult eNOS-KOs, in association with fewer native collaterals in hindlimb (confirmed in brain). This was not attributable to impaired collateral formation in the embryo-neonate, but rather from collateral loss during growth to adulthood. Compared to wild-type, eNOS-KOs evidenced reduced collateral remodeling, angiogenesis, and flow-mediated dilation of the arterial bed supplying the collaterals, resulting in lower perfusion and greater ischemic injury at all time points over 21 days following FAL. To probe the mechanism for impaired remodeling, we performed genome-wide expression profiling of isolated, remodeling hindlimb collaterals 24 hour after FAL. Upregulation of genes encoding cytokines/chemokines, inflammatory, stress response, and cell cycle proteins was evident in wild-type mice. In contrast, expression was lower in 40 of 44 cell cycle genes in eNOS-KO mice, in association with impaired proliferation of vascular wall cells. Conclusions: Our findings suggest a novel role for eNOS in maintaining native collateral density during natural growth to adulthood and in collateral remodeling in obstructive disease, the latter through regulation of cell proliferation.

Interaction between microvascular alpha 1- and alpha 2-adrenoceptors and endothelium-derived relaxing factor.

Intravital microscopy was used to study the effect of endothelium-derived relaxing factor (EDRF) on microvascular adrenoceptor sensitivity in rat cremaster skeletal muscle. NG-Monomethyl L-arginine (L-NMMA, 1-300 microM), an inhibitor of EDRF formation, produced concentration-dependent constriction of arterioles and venules. When an intermediate amount of alpha 1- versus alpha 2-adrenoceptor tone was first produced with bath-added norepinephrine (NE) in the presence of rauwolscine or prazosin, L-NMMA caused constriction with greater potency and efficacy during alpha 2 than during alpha 1 tone. During localized alpha 1 or alpha 2 constriction along an arteriole by perivascular micropipette suffusion of NE in the presence of rauwolscine or prazosin, again, bath-added L-NMMA produced constriction with greater potency during alpha 2 than during alpha 1 constriction. Like L-NMMA, disruption of EDRF release by microembolization caused baseline arteriole constriction and selectively increased alpha 2 sensitivity 75-fold. Although these findings support the hypothesis that endothelial cells possess alpha 2-adrenoceptors that promote EDRF release, a greater susceptibility of alpha 2 than alpha 1 constriction to EDRF inhibition could also account for the results. In support of this latter possibility, alpha 2 constriction was approximately 50-fold more susceptible than alpha 1 constriction to inhibition by the EDRF-like nitrodilator nitroprusside. The similarity in magnitude of this difference in sensitivity with the difference obtained in the embolization experiments does not support the hypothesis that microvascular endothelial cells in skeletal muscle possess EDRF-promoting alpha 2-adrenoceptors. However, these data do suggest that endogenous EDRF release modulates basal arteriole and venule tone and that alpha 2-adrenoceptor constriction is more sensitive than alpha 1 constriction to inhibition by EDRF.