Endothelial NOX5 overexpression induces changes in the cardiac gene profile: potential impact in myocardial infarction?

Cardiovascular diseases and the ischemic heart disease specifically constitute the main cause of death worldwide. The ischemic heart disease may lead to myocardial infarction, which in turn triggers numerous mechanisms and pathways involved in cardiac repair and remodeling. Our goal in the present study was to characterize the effect of the NADPH oxidase 5 (NOX5) endothelial expression in healthy and infarcted knock-in mice on diverse signaling pathways. The mechanisms studied in the heart of mice were the redox pathway, metalloproteinases and collagen pathway, signaling factors such as NFκB, AKT or Bcl-2, and adhesion molecules among others. Recent studies support that NOX5 expression in animal models can modify the environment and predisposes organ response to harmful stimuli prior to pathological processes. We found many alterations in the mRNA expression of components involved in cardiac fibrosis as collagen type I or TGF-β and in key players of cardiac apoptosis such as AKT, Bcl-2, or p53. In the heart of NOX5-expressing mice after chronic myocardial infarction, gene alterations were predominant in the redox pathway (NOX2, NOX4, p22phox, or SOD1), but we also found alterations in VCAM-1 and β-MHC expression. Our results suggest that NOX5 endothelial expression in mice preconditions the heart, and we propose that NOX5 has a cardioprotective role. The correlation studies performed between echocardiographic parameters and cardiac mRNA expression supported NOX5 protective action. (AU)

Endothelial NOX5 overexpression induces changes in the cardiac gene profile: potential impact in myocardial infarction?

Cardiovascular diseases and the ischemic heart disease specifically constitute the main cause of death worldwide. The ischemic heart disease may lead to myocardial infarction, which in turn triggers numerous mechanisms and pathways involved in cardiac repair and remodeling. Our goal in the present study was to characterize the effect of the NADPH oxidase 5 (NOX5) endothelial expression in healthy and infarcted knock-in mice on diverse signaling pathways. The mechanisms studied in the heart of mice were the redox pathway, metalloproteinases and collagen pathway, signaling factors such as NFκB, AKT or Bcl-2, and adhesion molecules among others. Recent studies support that NOX5 expression in animal models can modify the environment and predisposes organ response to harmful stimuli prior to pathological processes. We found many alterations in the mRNA expression of components involved in cardiac fibrosis as collagen type I or TGF-ß and in key players of cardiac apoptosis such as AKT, Bcl-2, or p53. In the heart of NOX5-expressing mice after chronic myocardial infarction, gene alterations were predominant in the redox pathway (NOX2, NOX4, p22phox, or SOD1), but we also found alterations in VCAM-1 and ß-MHC expression. Our results suggest that NOX5 endothelial expression in mice preconditions the heart, and we propose that NOX5 has a cardioprotective role. The correlation studies performed between echocardiographic parameters and cardiac mRNA expression supported NOX5 protective action.

Differential remodeling of subcutaneous white and interscapular brown adipose tissue by long-term exercise training in aged obese female mice.

Obesity exacerbates aging-induced adipose tissue dysfunction. This study aimed to investigate the effects of long-term exercise on inguinal white adipose tissue (iWAT) and interscapular brown adipose tissue (iBAT) of aged obese mice. Two-month-old female mice received a high-fat diet for 4 months. Then, six-month-old diet-induced obese animals were allocated to sedentarism (DIO) or to a long-term treadmill training (DIOEX) up to 18 months of age. In exercised mice, iWAT depot revealed more adaptability, with an increase in the expression of fatty acid oxidation genes (Cpt1a, Acox1), and an amelioration of the inflammatory status, with a favorable modulation of pro/antiinflammatory genes and lower macrophage infiltration. Additionally, iWAT of trained animals showed an increment in the expression of mitochondrial biogenesis (Pgc1a, Tfam, Nrf1), thermogenesis (Ucp1), and beige adipocytes genes (Cd137, Tbx1). In contrast, iBAT of aged obese mice was less responsive to exercise. Indeed, although an increase in functional brown adipocytes genes and proteins (Pgc1a, Prdm16 and UCP1) was observed, few changes were found on inflammation-related and fatty acid metabolism genes. The remodeling of iWAT and iBAT depots occurred along with an improvement in the HOMA index for insulin resistance and in glucose tolerance. In conclusion, long-term exercise effectively prevented the loss of iWAT and iBAT thermogenic properties during aging and obesity. In iWAT, the long-term exercise program also reduced the inflammatory status and stimulated a fat-oxidative gene profile. These exercise-induced adipose tissue adaptations could contribute to the beneficial effects on glucose homeostasis in aged obese mice.

Differential remodeling of subcutaneous white and interscapular brown adipose tissue by long-term exercise training in aged obese female mice

Obesity exacerbates aging-induced adipose tissue dysfunction. This study aimed to investigate the effects of long-term exercise on inguinal white adipose tissue (iWAT) and interscapular brown adipose tissue (iBAT) of aged obese mice. Two-month-old female mice received a high-fat diet for 4 months. Then, six-month-old diet-induced obese animals were allocated to sedentarism (DIO) or to a long-term treadmill training (DIOEX) up to 18 months of age. In exercised mice, iWAT depot revealed more adaptability, with an increase in the expression of fatty acid oxidation genes (Cpt1a, Acox1), and an amelioration of the inflammatory status, with a favorable modulation of pro/antiinflammatory genes and lower macrophage infiltration. Additionally, iWAT of trained animals showed an increment in the expression of mitochondrial biogenesis (Pgc1a, Tfam, Nrf1), thermogenesis (Ucp1), and beige adipocytes genes (Cd137, Tbx1). In contrast, iBAT of aged obese mice was less responsive to exercise. Indeed, although an increase in functional brown adipocytes genes and proteins (Pgc1a, Prdm16 and UCP1) was observed, few changes were found on inflammation-related and fatty acid metabolism genes. The remodeling of iWAT and iBAT depots occurred along with an improvement in the HOMA index for insulin resistance and in glucose tolerance. In conclusion, long-term exercise effectively prevented the loss of iWAT and iBAT thermogenic properties during aging and obesity. In iWAT, the long-term exercise program also reduced the inflammatory status and stimulated a fat-oxidative gene profile. These exercise-induced adipose tissue adaptations could contribute to the beneficial effects on glucose homeostasis in aged obese mice. (AU)

Adeno-Associated Viral Vectors as Versatile Tools for Neurological Disorders: Focus on Delivery Routes and Therapeutic Perspectives.

It is without doubt that the gene therapy field is currently in the spotlight for the development of new therapeutics targeting unmet medical needs. Thus, considering the gene therapy scenario, neurological diseases in general and neurodegenerative disorders in particular are emerging as the most appealing choices for new therapeutic arrivals intended to slow down, stop, or even revert the natural progressive course that characterizes most of these devastating neurodegenerative processes. Since an extensive coverage of all available literature is not feasible in practical terms, here emphasis was made in providing some advice to beginners in the field with a narrow focus on elucidating the best delivery route available for fulfilling any given AAV-based therapeutic approach. Furthermore, it is worth nothing that the number of ongoing clinical trials is increasing at a breath-taking speed. Accordingly, a landscape view of preclinical and clinical initiatives is also provided here in an attempt to best illustrate what is ongoing in this quickly expanding field.

Expression of Endothelial NOX5 Alters the Integrity of the Blood-Brain Barrier and Causes Loss of Memory in Aging Mice.

Blood-Brain barrier (BBB) disruption is a hallmark of central nervous system (CNS) dysfunction, and oxidative stress is one of the molecular mechanisms that may underlie this process. NADPH oxidases (NOX) are involved in oxidative stress-mediated vascular dysfunction and participate in the pathophysiology of its target organs. The NADPH oxidase 5 (NOX5) isoform is absent in rodents, and although little is known about the role it may play in disrupting the BBB, it has recently been implicated in experimental stroke. Our aim was to investigate the role of NADPH oxidase 5 (NOX5) in promoting vascular alterations and to identify its impact on the cognitive status of aged mice. No differences were detected in the arterial blood pressure or body weight between knock-in mice expressing endothelial NOX5 and the control mice. The Morris water maze test showed memory impairments in the aged knock-in mice expressing NOX5 compared with their control littermates. For assessing the BBB integrity, we studied the protein expression of two tight junction (TJ) proteins: Zonula occludens-1 (ZO-1) and occludin. Compared to the control animals, Aged NOX5 mice exhibited reduced levels of both proteins, demonstrating an alteration of the BBB integrity. Our data indicate that vascular NOX5 may favor behavioral changes with aging through oxidative stress-mediated BBB breakdown.

Adeno-Associated Viral Vectors as Versatile Tools for Parkinson's Research, Both for Disease Modeling Purposes and for Therapeutic Uses.

It is without any doubt that precision medicine therapeutic strategies targeting neurodegenerative disorders are currently witnessing the spectacular rise of newly designed approaches based on the use of viral vectors as Trojan horses for the controlled release of a given genetic payload. Among the different types of viral vectors, adeno-associated viruses (AAVs) rank as the ones most commonly used for the purposes of either disease modeling or for therapeutic strategies. Here, we reviewed the current literature dealing with the use of AAVs within the field of Parkinson's disease with the aim to provide neuroscientists with the advice and background required when facing a choice on which AAV might be best suited for addressing a given experimental challenge. Accordingly, here we will be summarizing some insights on different AAV serotypes, and which would be the most appropriate AAV delivery route. Next, the use of AAVs for modeling synucleinopathies is highlighted, providing potential readers with a landscape view of ongoing pre-clinical and clinical initiatives pushing forward AAV-based therapeutic approaches for Parkinson's disease and related synucleinopathies.

NADPH Oxidase 5 Induces Changes in the Unfolded Protein Response in Human Aortic Endothelial Cells and in Endothelial-Specific Knock-in Mice.

Oxidative stress constitutes a key molecular mechanism in the development of cardiovascular diseases. A potential relationship between reactive oxygen species (ROS) driven by the NADPH oxidase family (NOX) and the unfolded protein response (UPR) has been postulated. Nevertheless, there is a lack of information about the crosstalk between NOX5 homologue and the UPR in a cardiovascular context. The main aim was to analyze NOX5-mediated ROS effects in the UPR and its importance in cardiovascular diseases. To this effect, we used an adenoviral NOX5-ß overexpression model in human aortic endothelial cells (HAEC) and a conditional endothelial NOX5 knock-in mouse. Using expression arrays, we investigated NOX5-induced genomic changes in HAEC. Compared with the control HAEC, 298 genes were differentially expressed. Gene ontology analysis revealed the activation of numerous cellular routes, the most relevant being the UPR pathway. Using real-time PCR and Western Blot experiments, we confirmed that NOX5 overexpression induced changes in the expression of the UPR components, which were associated with increased apoptosis. Moreover, in endothelial-specific NOX5 knock-in mice, we found changes in the expression of the UPR components genes. In these mice, myocardial infarction was performed by permanent coronary artery ligation; however, NOX5 expression was not associated with differences in the UPR components mRNA levels. In these animals, we found significant associations between the UPR components gene expression and echocardiographic parameters. Our data support the idea that NOX5-derived ROS may modulate the UPR pathway in endothelial cells, which might play a relevant role in cardiac physiology.

Implications of NADPH oxidase 5 in vascular diseases.

Oxidative stress is one of the main mechanisms involved in the pathophysiology of vascular diseases. Among others, oxidative stress promotes endothelial dysfunction, and accelerated ageing and remodelling of vasculature. Lately, NADPH oxidases have been demonstrated to be involved in cardiovascular diseases. NADPH oxidase 5 has emerged as a new player in oxidative stress-mediated endothelial alterations, involved in the pathophysiology of hypertension, diabetes, atherosclerosis, myocardial infarction and stroke. This oxidase seems to mediate its detrimental effects by promoting inflammation. NADPH oxidase 5 has been studied in a lesser extent compared with the other members of the NADPH oxidase family due to its loss in the rodent genome, the main experimental research model. In addition, its potential as a therapeutic target remains unexplored given the lack of specific inhibitors. In this review the latest findings on NADPH oxidase 5 regulation, implications in vascular pathophysiology and therapeutic approaches will be updated.

Induction of Cyclooxygenase-2 by Overexpression of the Human NADPH Oxidase 5 (NOX5) Gene in Aortic Endothelial Cells.

Oxidative stress is a main molecular mechanism that underlies cardiovascular diseases. A close relationship between reactive oxygen species (ROS) derived from NADPH oxidase (NOX) activity and the prostaglandin (PG) biosynthesis pathway has been described. However, little information is available about the interaction between NOX5 homolog-derived ROS and the PG pathway in the cardiovascular context. Our main goal was to characterize NOX5-derived ROS effects in PG homeostasis and their potential relevance in cardiovascular pathologies. For that purpose, two experimental systems were employed: an adenoviral NOX5-ß overexpression model in immortalized human aortic endothelial cells (TeloHAEC) and a chronic infarction in vivo model developed from a conditional endothelial NOX5 knock-in mouse. NOX5 increased cyclooxygenase-2 isoform (COX-2) expression and prostaglandin E2 (PGE2) production through nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) in TeloHAEC. Protein kinase C (PKC) activation and intracellular calcium level (Ca++) mobilization increased ROS production and NOX5 overexpression, which promoted a COX-2/PGE2 response in vitro. In the chronic infarction model, mice encoding endothelial NOX5 enhanced the cardiac mRNA expression of COX-2 and PGES, suggesting a COX-2/PGE2 response to NOX5 presence in an ischemic situation. Our data support that NOX5-derived ROS may modulate the COX-2/PGE2 axis in endothelial cells, which might play a relevant role in the pathophysiology of heart infarction.

NADPH Oxidase Overactivity Underlies Telomere Shortening in Human Atherosclerosis.

Telomere shortening and oxidative stress are involved in the pathogenesis of atherosclerosis. Different studies have shown that phagocytic NADPH oxidase is associated with this disease. This study aimed to investigate the association between phagocytic NADPH oxidase and telomere shortening in human atherosclerosis. To assess this potential association, telomere length and phagocytic NADPH oxidase activity were determined by PCR and chemiluminescence, respectively, in a population of asymptomatic subjects free of overt clinical atherosclerosis. We also measured serum 8-hydroxy-2-deoxyguanosine (8-OHdG) levels (an index of oxidative stress) and carotid intima-media thickness (IMT), a surrogate marker of atherosclerosis. After adjusting them for age and sex, telomere length inversely correlated (p < 0.05) with NADPH oxidase-mediated superoxide production, with 8-OHdG values, and with carotid IMT. Interestingly, the asymptomatic subjects with plaques have a lower telomere length (p < 0.05), and higher values of plasma 8-OHdG and superoxide production (p < 0.05). These data were confirmed in a second population in which patients with coronary artery disease showed lower telomere length and higher 8-OHdG and superoxide production than the asymptomatic subjects. In both studies, NADPH oxidase-dependent superoxide production in phagocytic cells was only due to the specific expression of the Nox2 isoform. In conclusion, these findings suggest that phagocytic NADPH oxidase may be involved in oxidative stress-mediated telomere shortening, and that this axis may be critically involved in human atherosclerosis.

ICOS Costimulation at the Tumor Site in Combination with CTLA-4 Blockade Therapy Elicits Strong Tumor Immunity.

Cytotoxic T lymphocyte-associated protein 4 (CTLA-4) blockade therapy is able to induce long-lasting antitumor responses in a fraction of cancer patients. Nonetheless, there is still room for improvement in the quest for new therapeutic combinations. ICOS costimulation has been underscored as a possible target to include with CTLA-4 blocking treatment. Herein, we describe an ICOS agonistic aptamer that potentiates T cell activation and induces stronger antitumor responses when locally injected at the tumor site in combination with anti-CTLA-4 antibody in different tumor models. Furthermore, ICOS agonistic aptamer was engineered as a bi-specific tumor-targeting aptamer to reach any disseminated tumor lesions after systemic injection. Treatment with the bi-specific aptamer in combination with CTLA-4 blockade showed strong antitumor immunity, even in a melanoma tumor model where CTLA-4 treatment alone did not display any significant therapeutic benefit. Thus, this work provides strong support for the development of combinatorial therapies involving anti-CTLA-4 blockade and ICOS agonist tumor-targeting agents.

Systemic messenger RNA as an etiological treatment for acute intermittent porphyria.

Acute intermittent porphyria (AIP) results from haploinsufficiency of porphobilinogen deaminase (PBGD), the third enzyme in the heme biosynthesis pathway. Patients with AIP have neurovisceral attacks associated with increased hepatic heme demand. Phenobarbital-challenged mice with AIP recapitulate the biochemical and clinical characteristics of patients with AIP, including hepatic overproduction of the potentially neurotoxic porphyrin precursors. Here we show that intravenous administration of human PBGD (hPBGD) mRNA (encoded by the gene HMBS) encapsulated in lipid nanoparticles induces dose-dependent protein expression in mouse hepatocytes, rapidly normalizing urine porphyrin precursor excretion in ongoing attacks. Furthermore, hPBGD mRNA protected against mitochondrial dysfunction, hypertension, pain and motor impairment. Repeat dosing in AIP mice showed sustained efficacy and therapeutic improvement without evidence of hepatotoxicity. Finally, multiple administrations to nonhuman primates confirmed safety and translatability. These data provide proof-of-concept for systemic hPBGD mRNA as a potential therapy for AIP.

Increased phagocytic NADPH oxidase activity associates with coronary artery calcification in asymptomatic men.

Vascular calcification is a common feature in atherosclerosis and associates with cardiovascular events. Oxidative stress may be involved in the pathogenesis of vascular calcification. Previous studies have shown that the phagocytic NADPH oxidase is associated with atherosclerosis. The objective of the present study was to investigate the association between phagocytic NADPH oxidase-mediated superoxide production and coronary artery calcium (CAC). NADPH oxidase-mediated superoxide production was determined by chemiluminescence and CAC by computed tomography in 159 asymptomatic men free of overt clinical atherosclerosis. Multivariate linear regression analyses were used to assess the relationship between CAC and NADPH oxidase-mediated superoxide production. Compared with individuals in the lowest score of CAC (= 0 Agatston units), those in the upper score (>400 Agatston units) showed higher superoxide production (p < 0.05). In correlation analysis, superoxide production positively (p < 0.01) correlated with CAC, which in multivariate analysis remained significant after adjusting for age, HDL-cholesterol, triglycerides, body mass index, smoking, arterial hypertension and diabetes mellitus. In conclusion, in a population of men without clinically overt atherosclerotic disease, increased NADPH oxidase-mediated superoxide production associated with enhanced CAC. Albeit descriptive, these findings suggest a potential involvement of phagocytic NADPH oxidase-mediated oxidative stress in CAC.

Association of phagocytic NADPH oxidase activity with hypertensive heart disease: a role for cardiotrophin-1?

Left ventricular hypertrophy (LVH) is an independent marker of mortality in hypertension. Although the mechanisms contributing to LVH are complex, inflammation and oxidative stress may favor its development. We analyzed the association of the phagocytic NADPH oxidase-mediated superoxide anion release and LVH in patients with essential hypertension and the role of cardiotrophin-1 (CT-1) and interleukin-6 (IL-6), cytokines implicated in cardiac growth. Blood pressure, echocardiography data, and serum CT-1 and IL-6 levels were obtained in 140 subjects: 18 normotensives without LVH, 42 hypertensives without LVH, and 80 hypertensives with LVH. The NADPH oxidase-dependent superoxide production was assessed by chemiluminescence in peripheral blood mononuclear cells. Peripheral blood mononuclear cells were stimulated with CT-1 in vitro. Superoxide anion production by peripheral blood mononuclear cells associated with LVH and correlated with the left ventricular mass index. Serum CT-1 and IL-6 levels, which associated with the left ventricular mass index, correlated with superoxide production. Serum CT-1 and IL-6 levels were correlated. CT-1 stimulated NADPH oxidase superoxide production in peripheral blood mononuclear cells, which resulted in an increased release of IL-6. Our results show that superoxide anion production by the phagocytic NADPH oxidase associates with hypertensive heart disease, being significantly enhanced in hypertensive patients with LVH. This may be attributable to the activation of the NADPH oxidase by CT-1 and the subsequent release of IL-6. The phagocytic NADPH oxidase may be a therapeutic target in hypertensive heart disease.