Non-Interferon-Dependent Role of STING Signaling in Pulmonary Hypertension.

BACKGROUND: Patients with constitutive activation of DNA-sensing pathway through stimulator of IFN (interferon) genes (STING), such as those with STING-associated vasculopathy with onset in infancy, develop pulmonary hypertension (PH). However, the role of STING signaling in general PH patients is heretofore undescribed. Here, we seek to investigate the role of STING in PH development. METHODS: STING expression in patient lung samples was examined. PH was induced in global STING-deficient mice and global type I IFN receptor 1-deficient mice using bleomycin or chronic hypoxia exposure. PH development was evaluated by right ventricular systolic pressure and Fulton index, with additional histological and flow cytometric analysis. VEGF (vascular endothelial growth factor) expression on murine immune cells was quantified and evaluated with multiplex and flow cytometry. Human myeloid-derived cells were differentiated from peripheral blood mononuclear cells and treated with either STING agonist or STING antagonist for evaluation of VEGF secretion. RESULTS: Global STING deficiency protects mice from PH development, and STING-associated PH seems independent of type I IFN signaling. Furthermore, a role for STING-VEGF signaling pathway in PH development was demonstrated, with altered VEGF secretion in murine pulmonary infiltrated myeloid cells in a STING-dependent manner. In addition, pharmacological manipulation of STING in human myeloid-derived cells supports in vivo findings. Finally, a potential role of STING-VEGF-mediated apoptosis in disease development and progression was illustrated, providing a roadmap toward potential therapeutic applications. CONCLUSIONS: Overall, these data provide concrete evidence of STING involvement in PH, establishing biological plausibility for STING-related therapies in PH treatment.

Microglial Cells Impact Gut Microbiota and Gut Pathology in Angiotensin II-Induced Hypertension.

RATIONALE: Increased microglial activation and neuroinflammation within autonomic brain regions have been implicated in sustained hypertension, and their inhibition by minocycline-an anti-inflammatory antibiotic-produces beneficial effects. These observations led us to propose a dysfunctional brain-gut communication hypothesis for hypertension. However, it has been difficult to reconcile whether an anti-inflammatory or antimicrobial action is the primary beneficial effect of minocycline in hypertension. Accordingly, we utilized chemically modified tetracycline-3 (CMT-3)-a derivative of tetracycline that has potent anti-inflammatory activity-to address this question. OBJECTIVE: Test the hypothesis that central administration of CMT-3 would inhibit microglial activation, attenuate neuroinflammation, alter selective gut microbial communities, protect the gut wall from developing hypertension-associated pathology, and attenuate hypertension. METHODS AND RESULTS: Rats were implanted with radiotelemetry devices for recording mean arterial pressure. Ang II (angiotensin II) was infused subcutaneously using osmotic mini-pumps to induce hypertension. Another osmotic mini-pump was surgically implanted to infuse CMT-3 intracerebroventricularly. Intracerebroventricular CMT- 3 infusion was also investigated in SHR (spontaneously hypertensive rats). Physiological, pathological, immunohistological parameters, and fecal microbiota were analyzed. Intracerebroventricular CMT-3 significantly inhibited Ang II-induced increases in number of microglia, their activation, and proinflammatory cytokines in the paraventricular nucleus of hypothalamus. Further, intracerebroventricular CMT-3 attenuated increased mean arterial pressure, normalized sympathetic activity, and left ventricular hypertrophy in Ang II rats, as well as in the SHR. Finally, CMT-3 beneficially restored certain gut microbial communities altered by Ang II and attenuated pathological alterations in gut wall. CONCLUSIONS: These observations demonstrate that inhibition of microglial activation alone was sufficient to induce significant antihypertensive effects. This was associated with unique changes in gut microbial communities and profound attenuation of gut pathology. They suggest, for the first time, a link between microglia and certain microbial communities that may have implications for treatment of hypertension.

Transcriptomic signature of gut microbiome-contacting cells in colon of spontaneously hypertensive rats.

Fecal matter transfer from hypertensive patients and animals into normotensive animals increases blood pressure, strengthening the evidence for gut-microbiota interactions in the control of blood pressure. However, cellular and molecular events involved in gut dysbiosis-associated hypertension remain poorly understood. Therefore, our objective in this study was to use gene expression profiling to characterize the gut epithelium layer in the colon in hypertension. We observed significant suppression of components of T cell receptor (TCR) signaling in the colonic epithelium of spontaneously hypertensive rats (SHR) when compared with Wistar Kyoto (WKY) normotensive rats. Western blot analysis confirmed lower expression of key proteins including T cell surface glycoprotein CD3 gamma chain (Cd3g) and lymphocyte cytosolic protein 2 (Lcp2). Furthermore, lower expression of cytokines and receptors responsible for lymphocyte proliferation, differentiation, and activation (e.g., Il12r, Il15ra, Il7, Il16, Tgfb1) was observed in the colonic epithelium of the SHR. Finally, Alpi and its product, intestinal alkaline phosphatase, primarily localized in the epithelial cells, were profoundly lower in the SHR. These observations demonstrate that the colonic epithelium undergoes functional changes linked to altered immune, barrier function, and dysbiosis in hypertension.

Pulmonary hypertension: Pathophysiology beyond the lung.

Pulmonary hypertension (PH) is classically considered a disease of pulmonary vasculature which has been the predominant target for drug development and PH therapy. Despite significant advancement in recent years in identification of new drug targets and innovative treatment strategies, the prognosis of PH remains poor, with median survival of 5 years. Recent studies have demonstrated involvement of neuroinflammation, altered autonomic and gastrointestinal functions and increased trafficking of bone marrow-derived cells in cardiopulmonary pathophysiology. This has led to the proposal that PH could be considered a systemic disease involving complex interactions among many organs. Our objectives in this review is to summarize evidence for the involvement of the brain, bone marrow and gut in PH pathophysiology. Then, to synthesize all evidence supporting a brain-gut-lung interaction hypothesis for consideration in PH pathophysiology and finally to summarize unanswered questions and future directions to move this novel concept forward. This forward-thinking view, if proven by further experiments, would provide new opportunities and novel targets for the control and treatment of PH.

Chemokine signaling axis between endothelial and myeloid cells regulates development of pulmonary hypertension associated with pulmonary fibrosis and hypoxia.

Pulmonary hypertension complicates the care of many patients with chronic lung diseases (defined as Group 3 pulmonary hypertension), yet the mechanisms that mediate the development of pulmonary vascular disease are not clearly defined. Despite being the most prevalent form of pulmonary hypertension, to date there is no approved treatment for patients with disease. Myeloid-derived suppressor cells (MDSCs) and endothelial cells in the lung express the chemokine receptor CXCR2, implicated in the evolution of both neoplastic and pulmonary vascular remodeling. However, precise cellular contribution to lung disease is unknown. Therefore, we used mice with tissue-specific deletion of CXCR2 to investigate the role of this receptor in Group 3 pulmonary hypertension. Deletion of CXCR2 in myeloid cells attenuated the recruitment of polymorphonuclear MDSCs to the lungs, inhibited vascular remodeling, and protected against pulmonary hypertension. Conversely, loss of CXCR2 in endothelial cells resulted in worsened vascular remodeling, associated with increased MDSC migratory capacity attributable to increased ligand availability, consistent with analyzed patient sample data. Taken together, these data suggest that CXCR2 regulates MDSC activation, informing potential therapeutic application of MDSC-targeted treatments.

ACE2 overexpression in corticotropin-releasing-hormone cells offers protection against pulmonary hypertension.

Background: Pulmonary hypertension (PH), characterized by elevated pulmonary pressure and right heart failure, is a systemic disease involving inappropriate sympathetic activation and an impaired gut-brain-lung axis. Global overexpression of angiotensin converting enzyme 2 (ACE2), a cardiopulmonary protective enzyme of the renin-angiotensin system, attenuates PH induced by chronic hypoxia. Neurons within the paraventricular nucleus of the hypothalamus (PVN) that synthesize corticotropin-releasing hormone (CRH) are activated by stressors, like hypoxia, and this activation augments sympathetic outflow to cardiovascular tissues. These data coupled with our observations that ACE2 overexpression in CRH cells (CRH-ACE2KI mice) decreases anxiety-like behavior via suppression of hypothalamic-pituitary-adrenal (HPA) axis activity by decreasing CRH synthesis, led us to hypothesize that selective ACE2 overexpression in CRH neurons would protect against hypoxia-induced PH. Methods: CRH-ACE2KI and WT male and female mice were exposed to chronic hypoxia (10%O2) or normoxia (21%O2) for 4 weeks in a ventilated chamber with continuous monitoring of oxygen and carbon dioxide concentrations (n = 7-10/group). Pulmonary hemodynamics were measured with Millar pressure catheters then tissues were collected for histological analyses. Results: Chronic hypoxia induced a significant increase (36.4%) in right ventricular (RV) systolic pressure (RVSP) in WT mice, which was not observed in CRH-ACE2KI mice. No significant differences in RVSP were observed between male and female mice in any of the groups. Conclusion: Overexpression of ACE2 in CRH cells was protective against hypoxia-induced PH. Since the majority of expression of CRH is in brain nuclei such as paraventricular nucleus of the hypothalamus (PVN) and/or central nucleus of the amygdala (CeA) these data indicate that the protective effects of ACE2 are, at least in part, centrally mediated. This contributes to the systemic nature of PH disease and that CRH neurons may play an important role in PH.

Defining the age-dependent and tissue-specific circadian transcriptome in male mice.

Cellular circadian clocks direct a daily transcriptional program that supports homeostasis and resilience. Emerging evidence has demonstrated age-associated changes in circadian functions. To define age-dependent changes at the systems level, we profile the circadian transcriptome in the hypothalamus, lung, heart, kidney, skeletal muscle, and adrenal gland in three age groups. We find age-dependent and tissue-specific clock output changes. Aging reduces the number of rhythmically expressed genes (REGs), indicative of weakened circadian control. REGs are enriched for the hallmarks of aging, adding another dimension to our understanding of aging. Analyzing differential gene expression within a tissue at four different times of day identifies distinct clusters of differentially expressed genes (DEGs). Increased variability of gene expression across the day is a common feature of aged tissues. This analysis extends the landscape for understanding aging and highlights the impact of aging on circadian clock function and temporal changes in gene expression.

Fecal matter transplant from Ace2 overexpressing mice counteracts chronic hypoxia-induced pulmonary hypertension.

Recent evidence suggests pulmonary hypertension (PH), a disease of the pulmonary vasculature actually has multiorgan pathophysiology and perhaps etiology. Herein, we demonstrated that fecal matter transplantation from angiotensin-converting enzyme 2 overexpressing mice counteracted the effects of chronic hypoxia to prevent pulmonary hypertension, neuroinflammation, and gut dysbiosis in wild type recipients.

Central Administration of Hydrogen Sulfide Donor NaHS Reduces Iba1-Positive Cells in the PVN and Attenuates Rodent Angiotensin II Hypertension.

Hydrogen sulfide (H2S) is a gaseous signaling molecule with neuromodulatory, anti-inflammatory, and anti-hypertensive effects. Here, we investigate whether chronic intracerebroventricular (ICV) infusion of sodium hydrosulfide (NaHS), an H2S donor, can alleviate angiotensin II (Ang II)-induced hypertension (HTN), improve autonomic function, and impact microglia in the paraventricular nucleus (PVN) of the hypothalamus, a brain region associated with autonomic control of blood pressure (BP) and neuroinflammation in HTN. Chronic delivery of Ang II (200 ng/kg/min, subcutaneous) for 4 weeks produced a typical increase in BP and sympathetic drive and elevated the number of ionized calcium binding adaptor molecule 1-positive (Iba1+) cells in the PVN of male, Sprague-Dawley rats. ICV co-infusion of NaHS (at 30 and/or 60 nmol/h) significantly attenuated these effects of Ang II. Ang II also increased the abundance of cecal Deltaproteobacteria and Desulfovibrionales, among others, which was prevented by ICV NaHS co-infusion at 30 and 60 nmol/h. We observed no differences in circulating H2S between the groups. Our results suggest that central H2S may alleviate rodent HTN independently from circulating H2S via effects on autonomic nervous system and PVN microglia.

Charge-storage mechanism of highly defective NiO nanostructures on carbon nanofibers in electrochemical supercapacitors.

An electrode composed of highly defective nickel oxide (NiO) nanostructures supported on carbon nanofibers (CNFs) and immersed in an Li+-based aqueous electrolyte is studied using Raman spectroscopy under dynamic polarization conditions to address the charge-storage phenomenon. By this operando technique, the formation of Li2SO4·H2O during the discharge process is verified. At the same time, we observed the phase transformation of NiO to NiOOH. The Ni(OH)2/NiOOH redox couple is responsible for the pseudocapacitive behavior with intercalation of cationic species in the different Ni structures. A 'substitutive solid-state redox reaction' is proposed to represent the amphoteric nature of the oxide, resulting in proton intercalation, while the insertion of Li+ occurs to a less extent. The electrode material exhibits outstanding stability with 98% coulombic efficiency after 10 000 charge-discharge cycles. The excellent electrode properties can be ascribed to a synergism between CNFs and NiO, where the carbon nanostructures ensured rapid electron transport from the hydrated nickel nanoparticles. The NiO@CNF composite material is a promising candidate for future applications in aqueous-based supercapacitors. DFT simulation elucidates that compressive stress and Ni-site displacement lead to a decrease up-to 3.5-fold on the electron density map located onto the Ni-atom, which promotes NiO/Ni(OH)2/NiOOH transition.

Pediatric COVID-19 patients in South Brazil show abundant viral mRNA and strong specific anti-viral responses.

COVID-19 manifests as a milder disease in children than adults, but the underlying mechanisms are not fully characterized. Here we assess the difference in cellular or humoral immune responses of pediatric and adult COVID-19 patients to see if these factors contribute to the severity dichotomy. Children's non-specific immune profile is dominated by naive lymphocytes and HLA-DRhighCX3CR1low dendritic cells; meanwhile, children show strong specific antibody and T cell responses for viral structural proteins, with their T cell responses differing from adults by having weaker CD8+TNF+ T cells responses to S peptide pool but stronger responses to N and M peptide pools. Finally, viral mRNA is more abundant in pediatric patients. Our data thus support a scenario in which SARS-CoV-2 infected children contribute to transmission yet are less susceptible to COVID-19 symptoms due to strong and differential responses to the virus.

Pulmonary arterial hypertension-associated changes in gut pathology and microbiota.

Emerging evidence implicates an interplay among multiple organs such as brain, vasculature, gut and lung in the development of established pulmonary arterial hypertension (PAH). This has led us to propose that activated microglia mediated-enhanced sympathetic activation contributes to PAH pathophysiology. Since enhanced sympathetic activity is observed in human PAH and the gut is highly innervated by sympathetic nerves that regulate its physiological functions, we hypothesized that PAH would be associated with gut pathophysiology. A monocrotaline rat model of PAH was utilized to investigate the link between gut pathology and PAH. Haemodynamics, histology, immunocytochemistry and 16S RNA gene sequencing were used to assess cardiopulmonary functions, gut pathology and gut microbial communities respectively. Monocrotaline treatment caused increased right ventricular systolic pressure, haemodynamics and pathological changes associated with PAH. PAH animals also showed profound gut pathology that included increased intestinal permeability, increased muscularis layer, decreased villi length and goblet cells. These changes in gut pathology were associated with alterations in microbial communities, some unique to PAH animals. Furthermore, enhanced gut-neural communication involving the paraventricular nucleus of the hypothalamus and increased sympathetic drive were observed. In conclusion, our data show the presence of gut pathology and distinct changes in gut microbiota and increased sympathetic activity in PAH. They suggest that dysfunctional gut-brain crosstalk could be critical in PAH and considered a future therapeutic target for PAH.

Gut Pathology and Its Rescue by ACE2 (Angiotensin-Converting Enzyme 2) in Hypoxia-Induced Pulmonary Hypertension.

Therapeutic advances for pulmonary hypertension (PH) have been incremental because of the focus on the pulmonary vasculature in PH pathology. Here, we evaluate the concept that PH is, rather, a systemic disorder involving interplay among multiorgan systems, including brain, gut, and lungs. Therefore, the objective of this study was to evaluate the hypothesis that PH is associated with a dysfunctional brain-gut-lung axis and that global overexpression of ACE2 (angiotensin-converting enzyme 2) rebalances this axis and protects against PH. ACE2 knockin and wild-type (WT; C57BL/6) mice were subjected to chronic hypoxia (10% FIO2) or room air for 4 weeks. Cardiopulmonary hemodynamics, histology, immunohistochemistry, and fecal 16S rRNA microbial gene analyses were evaluated. Hypoxia significantly increased right ventricular systolic pressure, sympathetic activity as well as the number and activation of microglia in the paraventricular nucleus of the hypothalamus in WT mice. This was associated with a significant increase in muscularis layer thickening and decreases in both villi length and goblet cells and altered gut microbiota. Global overexpression of ACE2 prevented changes in hypoxia-induced pulmonary and gut pathophysiology and established distinct microbial communities from WT hypoxia mice. Furthermore, WT mice subjected to fecal matter transfer from ACE2 knockin mice were resistant to hypoxia-induced PH compared with their controls receiving WT fecal matter transfer. These observations demonstrate that ACE2 ameliorates these hypoxia-induced pathologies and attenuates PH. The data implicate dysfunctional brain-gut-lung communication in PH and provide novel avenues for therapeutic interventions.

Involvement of Neuroinflammation in the Pathogenesis of Monocrotaline-Induced Pulmonary Hypertension.

Pulmonary hypertension (PH) is a devastating disease and its successful treatment remains to be accomplished despite recent advances in pharmacotherapy. It has been proposed that PH be considered as a systemic disease, rather than primarily a disease of the pulmonary vasculature. Consequently, an investigation of the intricate interplay between multiple organs such as brain, vasculature, and lung in PH could lead to the identification of new targets for its therapy. However, little is known about this interplay. This study was undertaken to examine the concept that altered autonomic-pulmonary communication is important in PH pathophysiology. Therefore, we hypothesize that activation of microglial cells in the paraventricular nucleus of hypothalamus and neuroinflammation is associated with increased sympathetic drive and pulmonary pathophysiology contributing to PH. We utilized the monocrotaline rat model for PH and intracerebroventricular administration of minocycline for inhibition of microglial cells activation to investigate this hypothesis. Hemodynamic, echocardiographic, histological, immunohistochemical, and confocal microscopic techniques assessed cardiac and pulmonary function and microglial cells. Monocrotaline treatment caused cardiac and pulmonary pathophysiology associated with PH. There were also increased activated microglial cells and mRNA for proinflammatory cytokines (IL [interleukin]-1ß, IL-6, and TNF [tumor necrosis factor]-α) in the paraventricular nucleus. Furthermore, increased sympathetic drive and plasma norepinephrine were observed in rats with PH. Intracerebroventricular infusion of minocycline inhibited all these parameters and significantly attenuated PH. These observations implicate a dysfunctional autonomic-lung communication in the development and progression of PH providing new therapeutic targets, such as neuroinflammation, for PH therapy.

Angiotensin-(1-7) Promotes Resolution of Eosinophilic Inflammation in an Experimental Model of Asthma.

Defective apoptosis of eosinophils, the main leukocyte in the pathogenesis of asthma, and delay in its removal lead to lung damage and loss of pulmonary function due to failure in the resolution of inflammation. Here, we investigated the ability of angiotensin-(1-7) [Ang-(1-7)], a pivotal peptide of the renin-angiotensin system, to promote resolution of an allergic lung inflammatory response. Balb/c mice were sensitized and challenged with ovalbumin and treated with Ang-(1-7) at the peak of the inflammatory process. Bronchoalveolar lavage (BAL) fluid and lungs were collected 24 h after treatment. Different lung lobes were processed for histology to evaluate inflammatory cell infiltration, airway and pulmonary remodeling, total collagen staining, and measurements of (i) collagen I and III mRNA expression by qRT-PCR; (ii) ERK1/2, IκB-α, and GATA3 protein levels by Western blotting; and (iii) eosinophilic peroxidase activity. Total number of inflammatory cells, proportion of apoptotic eosinophils and immunofluorescence for caspase 3 and NF-κB in leukocytes were evaluated in the BAL. Mas receptor immunostaining was evaluated in mouse and human eosinophils. Engulfment of human polimorphonuclear cells by macrophages, efferocytosis, was evaluated in vivo. Ang-(1-7) reduced eosinophils in the lung and in the BAL, increased the number of apoptotic eosinophils, shown by histology criteria and by increase in caspase 3 immunostaining. Furthermore, Ang-(1-7) decreased NF-kB immunostaining in eosinophils, reduced GATA3, ERK1/2, and IκB-α expression in the lung and decreased pulmonary remodeling and collagen deposition. Importantly, Ang-(1-7) increased efferocytosis. Our results demonstrate, for the first time, Ang-(1-7) activates events that are crucial for resolution of the inflammatory process of asthma and promotion of the return of lung homeostasis, indicating Ang-(1-7) as novel endogenous inflammation-resolving mediator.

Comparison of multivariate calibration methods to determine simultaneously mebendazole-cambendazole and mebendazole-thiabendazole in pharmaceutical preparations by UV-visible spectrophotometry.

Three multivariate calibration methods, Principal Component Regression (PCR), the K-matrix method and Q-mode factor analysis followed by varimax and Imbrie's oblique rotations were applied to the simultaneous spectrophotometric determinations of mebendazole (MBZ)-cambendazole (CBZ) and thiabendazole (TBZ)-mebendazole in commercial samples of Exelmin and Helmiben. The calibration set concentrations were selected to contain a +/-10% variation in the quantity of active ingredients as declared by the manufacturer. The Q-mode factor analysis provides superior results for the two pharmaceutical formulations. The K-matrix method proved to be totally inadequate for these determinations. Almost all Q-mode results have relative errors much smaller than 5% of the active ingredient contents. This investigation shows that PCR and Q-mode factor analysis can be used to determine MBZ-CBZ and TBZ-MBZ in commercial drugs.