Advanced Magnetic Resonance Imaging to Define the Microvascular Injury Driven by Neuroinflammation in the Brain of a Mouse Model of Hypertension.

BACKGROUND: Hypertension is one of the main risk factors for dementia and cognitive impairment. METHODS: We used the model of transverse aortic constriction to induce chronic pressure overload in mice. We characterized brain injury by advanced translational applications of magnetic resonance imaging. In parallel, we analyzed peripheral target organ damage induced by chronic pressure overload by ultrasonography. Microscopical characterization of brain vasculature was performed as well, together with the analysis of immune and inflammatory markers. RESULTS: We identified a specific structural, microstructural, and functional brain injury. In particular, we highlighted a regional enlargement of the hypothalamus, microstructural damage in the white matter of the fimbria, and a reduction of the cerebral blood flow. A parallel analysis performed by confocal microscopy revealed a correspondent tissue damage evidenced by a reduction of cerebral capillary density, paired with loss of pericyte coverage. We assessed cognitive impairment and cardiac damage induced by hypertension to perform correlation analyses with the brain injury severity. At the mechanistic level, we found that CD8+T cells, producing interferon-γ, infiltrated the brain of hypertensive mice. By neutralizing this proinflammatory cytokine, we obtained a rescue of the phenotype, demonstrating their crucial role in establishing the microvascular damage. CONCLUSIONS: Overall, we have used translational tools to comprehensively characterize brain injury in a mouse model of hypertension induced by chronic pressure overload. We have identified early cerebrovascular damage in hypertensive mice, sustained by CD8+IFN-γ+T lymphocytes, which fuel neuroinflammation to establish the injury of brain capillaries.

Celiac Vagus Nerve Stimulation Recapitulates Angiotensin II-Induced Splenic Noradrenergic Activation, Driving Egress of CD8 Effector Cells.

Angiotensin II (AngII) is a peptide hormone that affects the cardiovascular system, not only through typical effects on the vasculature, kidneys, and heart, but also through less understood roles mediated by the brain and the immune system. Here, we address the hard-wired neural connections within the autonomic nervous system that modulate splenic immunity. Chronic AngII infusion triggers burst firing of the vagus nerve celiac efferent, an effect correlated with noradrenergic activation in the spleen and T cell egress. Bioelectronic stimulation of the celiac vagus nerve, in the absence of other challenges and independently from afferent signals to the brain, evokes the noradrenergic splenic pathway to promote release of a growth factor mediating neuroimmune crosstalk, placental growth factor (PlGF), and egress of CD8 effector T cells. Our findings also indicate that the neuroimmune interface mediated by PlGF and necessary for transducing the neural signal into an effective immune response is dependent on α-adrenergic receptor signaling.

Loss of EMILIN-1 Enhances Arteriolar Myogenic Tone Through TGF-ß (Transforming Growth Factor-ß)-Dependent Transactivation of EGFR (Epidermal Growth Factor Receptor) and Is Relevant for Hypertension in Mice and Humans.

Objective- EMILIN-1 (elastin microfibrils interface located protein-1) protein inhibits pro-TGF-ß (transforming growth factor-ß) proteolysis and limits TGF-ß bioavailability in vascular extracellular matrix. Emilin1-/- null mice display increased vascular TGF-ß signaling and are hypertensive. Because EMILIN-1 is expressed in vessels from embryonic life to adulthood, we aimed at unravelling whether the hypertensive phenotype of Emilin1-/- null mice results from a developmental defect or lack of homeostatic role in the adult. Approach and Results- By using a conditional gene targeting inactivating EMILIN-1 in smooth muscle cells of adult mice, we show that increased blood pressure in mice with selective smooth muscle cell ablation of EMILIN-1 depends on enhanced myogenic tone. Mechanistically, we unveil that higher TGF-ß signaling in smooth muscle cells stimulates HB-EGF (heparin-binding epidermal growth factor) expression and subsequent transactivation of EGFR (epidermal growth factor receptor). With increasing intraluminal pressure in resistance arteries, the cross talk established by TGF-ß and EGFR signals recruits TRPC6 (TRP [transient receptor potential] classical type 6) and TRPM4 (TRP melastatin type 4) channels, lastly stimulating voltage-dependent calcium channels and potentiating myogenic tone. We found reduced EMILIN-1 and enhanced myogenic tone, dependent on increased TGF-ß-EGFR signaling, in resistance arteries from hypertensive patients. Conclusions- Taken together, our findings implicate an unexpected role of the TGF-ß-EGFR pathway in hypertension with current translational perspectives.

Deoxycorticosterone acetate-salt hypertension activates placental growth factor in the spleen to couple sympathetic drive and immune system activation.

AIMS: Chronic increase of mineralocorticoids obtained by administration of deoxycorticosterone acetate (DOCA) results in salt-dependent hypertension in animals. Despite the lack of a generalized sympathoexcitation, DOCA-salt hypertension has been also associated to overdrive of peripheral nervous system in organs typically targeted by blood pressure (BP), as kidneys and vasculature. Aim of this study was to explore whether DOCA-salt recruits immune system by overactivating sympathetic nervous system in lymphoid organs and whether this is relevant for hypertension. METHODS AND RESULTS: To evaluate the role of the neurosplenic sympathetic drive in DOCA-salt hypertension, we challenged splenectomized mice or mice with left coeliac ganglionectomy with DOCA-salt, observing that they were both unable to increase BP. Then, we evaluated by immunofluorescence and ELISA levels of the placental growth factor (PlGF) upon DOCA-salt challenge, which significantly increased the growth factor expression, but only in the presence of an intact neurosplenic sympathetic drive. When PlGF KO mice were subjected to DOCA-salt, they were significantly protected from the increased BP observed in WT mice under same experimental conditions. In addition, absence of PlGF hampered DOCA-salt mediated T cells co-stimulation and their consequent deployment towards kidneys where they infiltrated tissue and provoked end-organ damage. CONCLUSION: Overall, our study demonstrates that DOCA-salt requires an intact sympathetic drive to the spleen for priming of immunity and consequent BP increase. The coupling of nervous system and immune cells activation in the splenic marginal zone is established through a sympathetic-mediated PlGF release, suggesting that this pathway could be a valid therapeutic target for hypertension.

Targeting Interleukin-1ß Protects from Aortic Aneurysms Induced by Disrupted Transforming Growth Factor ß Signaling.

Aortic aneurysms are life-threatening conditions with effective treatments mainly limited to emergency surgery or trans-arterial endovascular stent grafts, thus calling for the identification of specific molecular targets. Genetic studies have highlighted controversial roles of transforming growth factor ß (TGF-ß) signaling in aneurysm development. Here, we report on aneurysms developing in adult mice after smooth muscle cell (SMC)-specific inactivation of Smad4, an intracellular transducer of TGF-ß. The results revealed that Smad4 inhibition activated interleukin-1ß (IL-1ß) in SMCs. This danger signal later recruited innate immunity in the adventitia through chemokine (C-C motif) ligand 2 (CCL2) and modified the mechanical properties of the aortic wall, thus favoring vessel dilation. SMC-specific Smad4 deletion in Il1r1- or Ccr2-null mice resulted in milder aortic pathology. A chronic treatment with anti-IL-1ß antibody effectively hampered aneurysm development. These findings identify a mechanistic target for controlling the progression of aneurysms with compromised TGF-ß signaling, such as those driven by SMAD4 mutations.

La inhibición de la PI3Kγ protege contra la miocardiopatía diabética en ratones / PI3Kγ inhibition protects against diabetic cardiomyopathy in mice

Introducción y objetivos: Las enfermedades cardiovasculares, incluida la miocardiopatía, son las principales complicaciones de la diabetes mellitus. Un conocimiento más profundo de los mecanismos moleculares que conducen a la miocardiopatía es crucial para el desarrollo de nuevos tratamientos. Se propuso la fosfoinosítido 3-cinasa gamma (PI3Kγ) como objetivo molecular contra la miocardiopatía diabética, dado el papel que desempeña la PI3Kγ en el remodelado cardiaco frente a la sobrecarga de presión. Dada la disponibilidad de un inhibidor farmacológico de este objetivo molecular, el GE21, se ha investigado la validez de nuestra hipótesis induciendo la diabetes a ratones con ablación genética de la PI3Kγ, o knock-in, para una PI3Kγ catalíticamente inactiva. Métodos: Se provocó la diabetes a los ratones empleando estreptozotocina. Se evaluó la función cardiaca mediante exámenes ecocardiográficos secuenciales, mientras que la fibrosis y la inflamación se evaluaron mediante examen histológico. Resultados: La diabetes mellitus indujo disfunción cardiaca en los ratones genéticamente intactos (wild-type). La disfunción sistólica se previno completamente y la diastólica parcialmente, tanto en ratones con PI3Kγ inactivada como ratones sin actividad de cinasa. La disfunción cardiaca se recuperó en la misma medida administrando el inhibidor de la PI3Kγ GE21, de manera dependiente de la dosis. Estas acciones de inhibición genética o farmacológica de la PI3Kγ se asociaron con una reducción de la fibrosis y la inflamación en el corazón diabético. Conclusiones: Nuestro estudio demuestra un papel fundamental de la PI3Kγ en la miocardiopatía diabética en ratones y el efecto beneficioso de la inhibición farmacológica de la PI3Kγ; destaca su potencial como estrategia prometedora para el tratamiento clínico de las complicaciones cardiacas de los pacientes diabéticos (AU)

Introduction and objectives: Cardiovascular diseases, including cardiomyopathy, are the major complications in diabetes. A deeper understanding of the molecular mechanisms leading to cardiomyopathy is critical for developing novel therapies. We proposed phosphoinositide3-kinase gamma (PI3Kγ) as a molecular target against diabetic cardiomyopathy, given the role of PI3Kγ in cardiac remodeling to pressure overload. Given the availability of a pharmacological inhibitor of this molecular target GE21, we tested the validity of our hypothesis by inducing diabetes in mice with genetic ablation of PI3Kγ or knock-in for a catalytically inactive PI3Kγ. Methods: Mice were made diabetic by streptozotocin. Cardiac function was assessed by serial echocardiographic analyses, while fibrosis and inflammation were evaluated by histological analysis. Results: Diabetes induced cardiac dysfunction in wild-type mice. Systolic dysfunction was completely prevented, and diastolic dysfunction was partially blocked, in both PI3Kγ knock-out and kinase-dead mice. Cardiac dysfunction was similarly rescued by administration of the PI3Kγ inhibitor GE21 in a dose-dependent manner. These actions of genetic and pharmacological PI3Kγ inhibition were associated with a decrease in inflammation and fibrosis in diabetic hearts. Conclusions: Our study demonstrates a fundamental role of PI3Kγ in diabetic cardiomyopathy in mice and the beneficial effect of pharmacological PI3Kγ inhibition, highlighting its potential as a promising strategy for clinical treatment of cardiac complications of diabetic patients (AU)

PI3Kγ Inhibition Protects Against Diabetic Cardiomyopathy in Mice.

INTRODUCTION AND OBJECTIVES: Cardiovascular diseases, including cardiomyopathy, are the major complications in diabetes. A deeper understanding of the molecular mechanisms leading to cardiomyopathy is critical for developing novel therapies. We proposed phosphoinositide3-kinase gamma (PI3Kγ) as a molecular target against diabetic cardiomyopathy, given the role of PI3Kγ in cardiac remodeling to pressure overload. Given the availability of a pharmacological inhibitor of this molecular target GE21, we tested the validity of our hypothesis by inducing diabetes in mice with genetic ablation of PI3Kγ or knock-in for a catalytically inactive PI3Kγ. METHODS: Mice were made diabetic by streptozotocin. Cardiac function was assessed by serial echocardiographic analyses, while fibrosis and inflammation were evaluated by histological analysis. RESULTS: Diabetes induced cardiac dysfunction in wild-type mice. Systolic dysfunction was completely prevented, and diastolic dysfunction was partially blocked, in both PI3Kγ knock-out and kinase-dead mice. Cardiac dysfunction was similarly rescued by administration of the PI3Kγ inhibitor GE21 in a dose-dependent manner. These actions of genetic and pharmacological PI3Kγ inhibition were associated with a decrease in inflammation and fibrosis in diabetic hearts. CONCLUSIONS: Our study demonstrates a fundamental role of PI3Kγ in diabetic cardiomyopathy in mice and the beneficial effect of pharmacological PI3Kγ inhibition, highlighting its potential as a promising strategy for clinical treatment of cardiac complications of diabetic patients.

A cholinergic-sympathetic pathway primes immunity in hypertension and mediates brain-to-spleen communication.

The crucial role of the immune system in hypertension is now widely recognized. We previously reported that hypertensive challenges couple the nervous drive with immune system activation, but the physiological and molecular mechanisms of this connection are unknown. Here, we show that hypertensive challenges activate splenic sympathetic nerve discharge to prime immune response. More specifically, a vagus-splenic nerve drive, mediated by nicotinic cholinergic receptors, links the brain and spleen. The sympathetic discharge induced by hypertensive stimuli was absent in both coeliac vagotomized mice and in mice lacking α7nAChR, a receptor typically expressed by peripheral ganglionic neurons. This cholinergic-sympathetic pathway is necessary for T cell activation and egression on hypertensive challenges. In addition, we show that selectively thermoablating the splenic nerve prevents T cell egression and protects against hypertension. This novel experimental procedure for selective splenic denervation suggests new clinical strategies for resistant hypertension.

Lack of kinase-independent activity of PI3Kγ in locus coeruleus induces ADHD symptoms through increased CREB signaling.

Although PI3Kγ has been extensively investigated in inflammatory and cardiovascular diseases, the exploration of its functions in the brain is just at dawning. It is known that PI3Kγ is present in neurons and that the lack of PI3Kγ in mice leads to impaired synaptic plasticity, suggestive of a role in behavioral flexibility. Several neuropsychiatric disorders, such as attention-deficit/hyperactivity disorder (ADHD), involve an impairment of behavioral flexibility. Here, we found a previously unreported expression of PI3Kγ throughout the noradrenergic neurons of the locus coeruleus (LC) in the brainstem, serving as a mechanism that regulates its activity of control on attention, locomotion and sociality. In particular, we show an unprecedented phenotype of PI3Kγ KO mice resembling ADHD symptoms. PI3Kγ KO mice exhibit deficits in the attentive and mnemonic domains, typical hyperactivity, as well as social dysfunctions. Moreover, we demonstrate that the ADHD phenotype depends on a dysregulation of CREB signaling exerted by a kinase-independent PI3Kγ-PDE4D interaction in the noradrenergic neurons of the locus coeruleus, thus uncovering new tools for mechanistic and therapeutic research in ADHD.

The angiogenic factor PlGF mediates a neuroimmune interaction in the spleen to allow the onset of hypertension.

Hypertension is a health problem affecting over 1 billion people worldwide. How the immune system gets activated under hypertensive stimuli to contribute to blood pressure elevation is a fascinating enigma. Here we showed a splenic role for placental growth factor (PlGF), which accounts for the onset of hypertension, through immune system modulation. PlGF repressed the expression of the protein Timp3 (tissue inhibitor of metalloproteinases 3), through the transcriptional Sirt1-p53 axis. Timp3 repression allowed costimulation of T cells and their deployment toward classical organs involved in hypertension. We showed that the spleen is an essential organ for the development of hypertension through a noradrenergic drive mediated by the celiac ganglion efferent. Overall, we demonstrate that PlGF mediates the neuroimmune interaction in the spleen, organizing a unique and nonredundant response that allows the onset of hypertension.