Giuseppe Faraco

Commensal microbiota affects ischemic stroke outcome by regulating intestinal γδ T cells

Commensal gut bacteria impact the host immune system and can influence disease processes in several organs, including the brain. However, it remains unclear whether the microbiota has an impact on the outcome of acute brain injury. Here we show that antibiotic-induced alterations in the intestinal flora reduce ischemic brain injury in mice, an effect transmissible by fecal transplants. Intestinal dysbiosis alters immune homeostasis in the small intestine, leading to an increase in regulatory T cells and a reduction in interleukin (IL)-17–positive γδ T cells through altered dendritic cell activity. Dysbiosis suppresses trafficking of effector T cells from the gut to the leptomeninges after stroke. Additionally, IL-10 and IL-17 are required for the neuroprotection afforded by intestinal dysbiosis. The findings reveal a previously unrecognized gut-brain axis and an impact of the intestinal flora and meningeal IL-17+ γδ T cells on ischemic injury.

Hypertension: A Harbinger of Stroke and Dementia

Despite remarkable successes in prevention and treatment, hypertension (HTN) remains a major cause of morbidity and mortality worldwide.1 The brain is one of the preeminent target organs in which high blood pressure is particularly damaging, contributing significantly to its burden of disease.1 HTN is the biggest risk factor for stroke, the second cause of death worldwide and a major cause of long-term disability.2 HTN is also a leading risk factor for vascular cognitive impairment (VCI),3 as well as Alzheimer disease (AD), the most common cause of dementia in the elderly.3 Therefore, HTN is involved in the pathogenesis of 2 major brain diseases: stroke and dementia. In this brief review, we first examine the role of HTN in the alterations in cerebrovascular structure and function underlying the pathological effects of HTN on the brain. Then, we examine the pathophysiological bases of these alterations, focusing on how high blood pressure promotes stroke and cognitive impairment. Finally, we discuss the impact of preventive and therapeutic approaches to mitigate the deleterious effects of HTN on the brain. ### Adaptive Changes: Hypertrophy and Remodeling Sustained elevations in blood pressure have profound effects on the structure of cerebral blood vessels, inducing adaptive changes aimed at reducing the mechanical stress on the arterial wall and protecting microvessels from pulsatile stress.4 In hypertrophic remodeling, the media thickens to encroach into the lumen, resulting in increased media cross-sectional area and media/lumen ratio (Figure 1). The size of vascular smooth muscle cells increases, and there is accumulation of extracellular matrix proteins, such as collagen and fibronectin, in the vessel wall.5 In eutrophic remodeling, smooth muscle cells undergo a rearrangement that leads to a reduction in the outer and inner diameters, whereas media/lumen ratio is increased and cross-sectional area is unaltered (Figure 1). In this case, the change in vascular …

Inhibition of Nicotinamide Phosphoribosyltransferase: CELLULAR BIOENERGETICS REVEALS A MITOCHONDRIAL INSENSITIVE NAD POOL*

The NAD rescue pathway consists of two enzymatic steps operated by nicotinamide phosphoribosyltransferase (Nampt) and nicotinamide mononucleotide adenylyltransferases. Recently, the potent Nampt inhibitor FK866 has been identified and evaluated in clinical trials against cancer. Yet, how Nampt inhibition affects NAD contents and bioenergetics is in part obscure. It is also unknown whether NAD rescue takes place in mitochondria, and FK866 alters NAD homeostasis within the organelle. Here, we show that FK866-dependent reduction of the NAD contents is paralleled by a concomitant increase of ATP in various cell types, in keeping with ATP utilization for NAD resynthesis. We also show that poly- and mono(ADP-ribose) transferases rather than Sirt-1 are responsible for NAD depletion in HeLa cells exposed to FK866. Mass spectrometry reveals that the drug distributes in the cytosolic and mitochondrial compartment. However, the cytoplasmic but not the mitochondrial NAD pool is reduced upon acute or chronic exposure to the drug. Accordingly, Nampt does not localize within the organelles and their bioenergetics is not affected by the drug. In the mouse, FK866-dependent reduction of NAD contents in various organs is prevented by inhibitors of poly(ADP-ribose) polymerases or the NAD precursor kynurenine. For the first time, our data indicate that mitochondria lack the canonical NAD rescue pathway, broadening current understanding of cellular bioenergetics.

Central Cardiovascular Circuits Contribute to the Neurovascular Dysfunction in Angiotensin II Hypertension

Hypertension, a powerful risk factor for stroke and dementia, has damaging effects on the brain and its vessels. In particular, hypertension alters vital cerebrovascular control mechanisms linking neural activity to cerebral perfusion. In experimental models of slow-developing hypertension, free radical signaling in the subfornical organ (SFO), one of the forebrain circumventricular organs, is critical for the hormonal release and sympathetic activation driving the elevation in arterial pressure. However, the contribution of this central mechanism to the cerebrovascular alterations induced by hypertension remains uncertain. We tested the hypothesis that free radical production in the SFO is involved in the alterations in cerebrovascular regulation produced by hypertension. In a mouse model of gradual hypertension induced by chronic administration of subpressor doses of angiotensin II (AngII), suppression of free radicals in the SFO by overexpression of CuZn-superoxide dismutase (CuZnSOD) prevented the alteration in neurovascular coupling and endothelium-dependent responses in somatosensory cortex induced by hypertension. The SFO mediates the dysfunction via two signaling pathways. One involves SFO-dependent activation of the paraventricular hypothalamic nucleus, elevations in plasma vasopressin, upregulation of endothelin-1 in cerebral resistance arterioles and activation of endothelin type A receptors. The other pathway depends on activation of cerebrovascular AngII type 1 (AT1) receptors by AngII. Both pathways mediate vasomotor dysfunction by inducing vascular oxidative stress. The findings implicate for the first time the SFO and its efferent hypothalamic pathways in the cerebrovascular alterations induced by AngII, and identify vasopressin and endothelin-1 as potential therapeutic targets to counteract the devastating effects of hypertension on the brain.

The Therapeutic Potential of HDAC Inhibitors in the Treatment of Multiple Sclerosis

Multiple sclerosis (MS) is a devastating autoimmune disorder of the central nervous system (CNS) for which there is no efficacious cure. Thanks to numerous preclinical and clinical studies, drugs able to mitigate the inexorable course of the disease have been made available recently. Still, there is a terrible need for compounds capable of reducing the severity of the autoimmune attack and of blocking progression of the disorder. Also, besides the classic immunosuppressive strategies, it is now appreciated that compounds directly targeting neuronal death can be of relevance to the treatment of MS patients. Acetylation homeostasis is a key regulator of both immune cell activation and neuronal survival. Of note, potent histone deacetylase inhibitors (HDACi) endowed with antiinflammatory and neuroprotective properties have been identified. Efficacy of HDACi in experimental models of MS has been reported consistently. In this review, we provide an appraisal of the literature on HDACi and MS, also discussing the mechanisms by which HDACi can suppress the autoimmune attack to the CNS.

Cyclooxygenase 1–Derived Prostaglandin E2 and EP1 Receptors Are Required for the Cerebrovascular Dysfunction Induced by Angiotensin II

Prostaglandin E2 (PGE2) EP1 receptors (EP1Rs) may contribute to hypertension and related end-organ damage. Because of the key role of angiotensin II (Ang II) in hypertension, we investigated the role of EP1R in the cerebrovascular alterations induced by Ang II. Mice were equipped with a cranial window, and cerebral blood flow was monitored by laser-Doppler flowmetry. The attenuation in cerebral blood flow responses to whisker stimulation (−46±4%) and the endothelium-dependent vasodilator acetylcholine (−40±4%) induced by acute administration of Ang II (250 ng/kg per minute; IV for 30 to 40 minutes) were not observed after cyclooxygenase 1 or EP1R inhibition or in cyclooxygenase 1 or EP1-null mice. In contrast, cyclooxygenase 2 inhibition or genetic inactivation did not prevent the attenuation. Ang II–induced oxidative stress was not observed after cyclooxygenase 1 or EP1R inhibition or in EP1R-null mice. Prostaglandin E2 reinstated the Ang II–induced cerebrovascular dysfunction and oxidative stress after cyclooxygenase 1 inhibition. Brain prostaglandin E2 levels were not increased by Ang II but were attenuated by cyclooxygenase 1 and not cyclooxygenase 2 inhibition. The cerebrovascular dysfunction induced by 14-day administration of “slow-pressor” doses of Ang II (600 ng/kg per minute) was attenuated by neocortical application of SC51089. Cyclooxygenase 1 immunoreactivity was observed in microglia and EP1R in endothelial cells. We conclude that the cerebrovascular dysfunction induced by Ang II requires activation of EP1R by constitutive production of prostaglandin E2 derived from cyclooxygenase 1. The findings provide the first evidence that EP1Rs are involved in the deleterious cerebrovascular effects of Ang II and suggest new therapeutic approaches to counteract them.

Perivascular macrophages mediate the neurovascular and cognitive dysfunction associated with hypertension

Hypertension is a leading risk factor for dementia, but the mechanisms underlying its damaging effects on the brain are poorly understood. Due to a lack of energy reserves, the brain relies on continuous delivery of blood flow to its active regions in accordance with their dynamic metabolic needs. Hypertension disrupts these vital regulatory mechanisms, leading to the neuronal dysfunction and damage underlying cognitive impairment. Elucidating the cellular bases of these impairments is essential for developing new therapies. Perivascular macrophages (PVMs) represent a distinct population of resident brain macrophages that serves key homeostatic roles but also has the potential to generate large amounts of reactive oxygen species (ROS). Here, we report that PVMs are critical in driving the alterations in neurovascular regulation and attendant cognitive impairment in mouse models of hypertension. This effect was mediated by an increase in blood-brain barrier permeability that allowed angiotensin II to enter the perivascular space and activate angiotensin type 1 receptors in PVMs, leading to production of ROS through the superoxide-producing enzyme NOX2. These findings unveil a pathogenic role of PVMs in the neurovascular and cognitive dysfunction associated with hypertension and identify these cells as a putative therapeutic target for diseases associated with cerebrovascular oxidative stress.

Hypertension enhances Aβ-induced neurovascular dysfunction, promotes β-secretase activity, and leads to amyloidogenic processing of APP

Hypertension (HTN) doubles the risk of Alzheimer’s disease (AD), but the mechanisms remain unclear. Amyloid-β (Aβ), a key pathogenic factor in AD, induces cerebrovascular dysfunction. We hypothesized that HTN acts in concert with Aβ to amplify its deleterious cerebrovascular effects and to increase Aβ production. Infusion of angiotensin II (ANGII; intravenously) elevated blood pressure and attenuated the cerebral blood flow (CBF) response to whisker stimulation or the endothelium-dependent vasodilator acetylcholine (ACh) (P < 0.05). Neocortical application of Aβ in mice receiving ANGII worsened the responses to ACh (P < 0.05). The cerebrovascular dysfunction observed in Tg2576 mice, in which Aβ is elevated both in blood and in brain due to expression of mutated amyloid precursor protein (APP), was not aggravated by neocortical application of ANGII or by a 2-week administration of ‘slow pressor’ of ANGII (600 ng/kg per minute; subcutaneously). In contrast, ANGII aggravated the dysfunction in TgSwDI mice, in which Aβ is increased only in brain. Slow-pressor ANGII induced microvascular amyloid deposition in Tg2576 mice and enhanced β-secretase APP cleavage. In Chinese hamster ovary (CHO) cells producing Aβ, ANGII increased β-secretase activity, Aβ1–42, and the Aβ42/40 ratio. We conclude that HTN enhances amyloidogenic APP processing, effects that may contribute to the pathogenic interaction between HTN and AD.

Endothelin 1–Dependent Neurovascular Dysfunction in Chronic Intermittent Hypoxia

Obstructive sleep apnea, a condition resulting in chronic intermittent hypoxia (CIH), is an independent risk factor for stroke and dementia, but the mechanisms of the effect are unknown. We tested the hypothesis that CIH increases cerebrovascular risk by altering critical mechanisms regulating cerebral blood flow thereby lowering cerebrovascular reserves. Male C57Bl6/J mice were subjected to CIH (10% O2 for 90 seconds/room air for 90 seconds; during sleep hours) or sham treatment for 35 days. Somatosensory cortex blood flow was assessed by laser Doppler flowmetry in anesthetized mice equipped with a cranial window. CIH increased mean arterial pressure (from 74±2 to 83±3 mm Hg; P<0.05) and attenuated the blood flow increase produced by neural activity (whisker stimulation; −39±2%; P<0.05) or neocortical application of endothelium-dependent vasodilators (acetylcholine response: −41±3%; P<0.05). The cerebrovascular dysfunction was associated with oxidative stress in cerebral resistance arterioles and was abrogated by free radical scavenging or NADPH oxidase inhibition. Furthermore, cerebrovascular dysfunction and free radical increase were not observed in mice lacking the NOX2 subunit of NADPH oxidase. CIH markedly increased endothelin 1 in cerebral blood vessels, whereas cerebrovascular dysfunction and oxidative stress were abrogated by neocortical application of the endothelin type A receptor antagonist BQ123. These data demonstrate for the first time that CIH alters key regulatory mechanisms of the cerebral circulation through endothelin 1 and NADPH oxidase–derived radicals. The ensuing cerebrovascular dysfunction may increase stroke risk in patients with sleep apnea by reducing cerebrovascular reserves and increasing the brains susceptibility to cerebral ischemia.

Circulating Endothelin-1 Alters Critical Mechanisms Regulating Cerebral Microcirculation

Endothelin-1 (ET1) is a potent vasoconstrictor peptide implicated in the cerebrovascular alterations occurring in stroke, subarachnoid hemorrhage, and brain trauma. Brain or circulating levels of ET1 are elevated in these conditions and in risk factors for cerebrovascular diseases. Most studies on the cerebrovascular effects of ET1 have focused on vascular smooth muscle constriction, and little is known about the effect of the peptide on cerebrovascular regulation. We tested the hypothesis that ET1 increases cerebrovascular risk by disrupting critical mechanisms regulating cerebral blood flow. Male C57Bl6/J mice equipped with a cranial window were infused intravenously with vehicle or ET1, and somatosensory cortex blood flow was assessed by laser Doppler flowmetry. ET1 infusion increased mean arterial pressure and attenuated the blood flow increase produced by neural activity (whisker stimulation) or neocortical application of the endothelium-dependent vasodilator acetylcholine but not A23187. The cerebrovascular effects of ET1 were abrogated by the ETA receptor antagonist BQ123 and were not related to vascular oxidative stress. Rather, the dysfunction was dependent on Rho-associated protein kinase activity. Furthermore, in vitro studies demonstrated that ET1 suppresses endothelial nitric oxide (NO) production, assessed by its metabolite nitrite, an effect associated with Rho-associated protein kinase–dependent changes in the phosphorylation state of endothelial NO synthase. Collectively, these novel observations demonstrate that increased ET1 plasma levels alter key regulatory mechanisms of the cerebral circulation by modulating endothelial NO synthase phosphorylation and NO production through Rho-associated protein kinase. The ET1-induced cerebrovascular dysfunction may increase cerebrovascular risk by lowering cerebrovascular reserves and increasing the vulnerability of the brain to cerebral ischemia.