Comparison of l-Carnitine and l-Carnitine HCL salt for targeted lung treatment of pulmonary hypertension (PH) as inhalation aerosols: Design, comprehensive characterization, in vitro 2D/3D cell cultures, and in vivo MCT-Rat model of PH.

Disrupted l-Carnitine (L-Car) homeostasis has been implicated in the development of pulmonary hypertension (PH). L-Car has been administered orally and intravenously causing systemic side effects. To the authors' knowledge, there are no reports using L-Car or L-Car HCl as an inhaled aerosol through the respiratory route in a targeted manner either from dry powder inhaler (DPI) or liquid delivery system. The purpose of the comprehensive and systematic comparative study between L-Car and L-Car HCl salt was to design and develop dry powder inhalers (DPIs) of each. This was followed by comprehensive physicochemical characterization, in vitro cell viability as a function of dose on 2D human pulmonary cell lines from different lung regions and in vitro cell viability on 3D small airway epithelia human primary cells at the air-liquid interface (ALI). In addition in vitro transepithelial electrical resistance (TEER) in air-interface culture (AIC) conditions on 2D human pulmonary cell line and 3D small airway epithelia human primary cells was carried out. In vitro aerosol dispersion performance using three FDA-approved human DPI devices with different device properties was also examined. Following advanced spray drying under various conditions, two spray drying pump rates (low and medium) were found to successfully produce spray-dried L-Car powders while four spray drying pump rates (low, medium, medium-high, and high) all resulted in the production of spray-dried L-Car HCl powders. Raw L-Car and L-Car HCl were found to be crystalline. All SD powders retained crystallinity following spray drying and polymorphic interconversion in the solid-state was identified as the mechanism for retaining crystallinity after the advanced spray drying process. All SD powders aerosolized readily with all three human DPI devices. However, the in vitro dispersion parameters for the SD powders was not conducive for in vivo administration to rats in DPIs due to hygroscopicity and nanoaggreation. In vivo rat studies were successfully accomplished using inhaled liquid aerosols. Safety was successfully demonstrated in vivo in healthy Sprague Dawley rats. Furthermore, therapeutic efficacy was successfully demonstrated in vivo in the monocrotaline (MCT)-rat model of PH after two weeks of daily L-Car inhalation aerosol treatment.

Biomechanical Forces and Oxidative Stress: Implications for Pulmonary Vascular Disease.

Significance: Oxidative stress in the cell is characterized by excessive generation of reactive oxygen species (ROS). Superoxide (O2-) and hydrogen peroxide (H2O2) are the main ROS involved in the regulation of cellular metabolism. As our fundamental understanding of the underlying causes of lung disease has increased it has become evident that oxidative stress plays a critical role. Recent Advances: A number of cells in the lung both produce, and respond to, ROS. These include vascular endothelial and smooth muscle cells, fibroblasts, and epithelial cells as well as the cells involved in the inflammatory response, including macrophages, neutrophils, eosinophils. The redox system is involved in multiple aspects of cell metabolism and cell homeostasis. Critical Issues: Dysregulation of the cellular redox system has consequential effects on cell signaling pathways that are intimately involved in disease progression. The lung is exposed to biomechanical forces (fluid shear stress, cyclic stretch, and pressure) due to the passage of blood through the pulmonary vessels and the distension of the lungs during the breathing cycle. Cells within the lung respond to these forces by activating signal transduction pathways that alter their redox state with both physiologic and pathologic consequences. Future Directions: Here, we will discuss the intimate relationship between biomechanical forces and redox signaling and its role in the development of pulmonary disease. An understanding of the molecular mechanisms induced by biomechanical forces in the pulmonary vasculature is necessary for the development of new therapeutic strategies.

Gut Microbiota Contributes to Resistance Against Pneumococcal Pneumonia in Immunodeficient Rag-/- Mice.

Streptococcus pneumoniae causes infection-related mortality worldwide. Immunocompromised individuals, including young children, the elderly, and those with immunodeficiency, are especially vulnerable, yet little is known regarding S. pneumoniae-related pathogenesis and protection in immunocompromised hosts. Recently, strong interest has emerged in the gut microbiota's impact on lung diseases, or the "gut-lung axis." However, the mechanisms of gut microbiota protection against gut-distal lung diseases like pneumonia remain unclear. We investigated the role of the gut commensal, segmented filamentous bacteria (SFB), against pneumococcal pneumonia in immunocompetent and immunocompromised mouse models. For the latter, we chose the Rag-/- model, with adaptive immune deficiency. Immunocompetent adaptive protection against S. pneumoniae infection is based on antibodies against pneumococcal capsular polysaccharides, prototypical T cell independent-II (TI-II) antigens. Although SFB colonization enhanced TI-II antibodies in C57BL/6 mice, our data suggest that SFB did not further protect these immunocompetent animals. Indeed, basal B cell activity in hosts without SFB is sufficient for essential protection against S. pneumoniae. However, in immunocompromised Rag-/- mice, we demonstrate a gut-lung axis of communication, as SFB influenced lung protection by regulating innate immunity. Neutrophil resolution is crucial to recovery, since an unchecked neutrophil response causes severe tissue damage. We found no early neutrophil recruitment differences between hosts with or without SFB; however, we observed a significant drop in lung neutrophils in the resolution phase of S. pneumoniae infection, which corresponded with lower CD47 expression, a molecule that inhibits phagocytosis of apoptotic cells, in SFB-colonized Rag-/- mice. SFB promoted a shift in lung neutrophil phenotype from inflammatory neutrophils expressing high levels of CD18 and low levels of CD62L, to pro-resolution neutrophils with low CD18 and high CD62L. Blocking CD47 in SFB(-) mice increased pro-resolution neutrophils, suggesting CD47 down-regulation may be one neutrophil-modulating mechanism SFB utilizes. The SFB-induced lung neutrophil phenotype remained similar with heat-inactivated S. pneumoniae treatment, indicating these SFB-induced changes in neutrophil phenotype during the resolution phase are not simply secondary to better bacterial clearance in SFB(+) than SFB(-) mice. Together, these data demonstrate that the gut commensal SFB may provide much-needed protection in immunocompromised hosts in part by promoting neutrophil resolution post lung infection.

LPS-induced Acute Lung Injury Involves NF-κB-mediated Downregulation of SOX18.

One of the early events in the progression of LPS-mediated acute lung injury in mice is the disruption of the pulmonary endothelial barrier resulting in lung edema. However, the molecular mechanisms by which the endothelial barrier becomes compromised remain unresolved. The SRY (sex-determining region on the Y chromosome)-related high-mobility group box (Sox) group F family member, SOX18, is a barrier-protective protein through its ability to increase the expression of the tight junction protein CLDN5. Thus, the purpose of this study was to determine if downregulation of the SOX18-CLDN5 axis plays a role in the pulmonary endothelial barrier disruption associated with LPS exposure. Our data indicate that both SOX18 and CLDN5 expression is decreased in two models of in vivo LPS exposure (intraperitoneal, intratracheal). A similar downregulation was observed in cultured human lung microvascular endothelial cells (HLMVECs) exposed to LPS. SOX18 overexpression in HLMVECs or in the mouse lung attenuated the LPS-mediated vascular barrier disruption. Conversely, reduced CLDN5 expression (siRNA) reduced the HLMVEC barrier-protective effects of SOX18 overexpression. The mechanism by which LPS decreases SOX18 expression was identified as transcriptional repression through binding of NF-κB (p65) to a SOX18 promoter sequence located between -1,082 and -1,073 bp with peroxynitrite contributing to LPS-mediated NF-κB activation. We conclude that NF-κB-dependent decreases in the SOX18-CLDN5 axis are essentially involved in the disruption of human endothelial cell barrier integrity associated with LPS-mediated acute lung injury.

Gut Microbiota Drive Autoimmune Arthritis by Promoting Differentiation and Migration of Peyer's Patch T Follicular Helper Cells.

Gut microbiota profoundly affect gut and systemic diseases, but the mechanism whereby microbiota affect systemic diseases is unclear. It is not known whether specific microbiota regulate T follicular helper (Tfh) cells, whose excessive responses can inflict antibody-mediated autoimmunity. Using the K/BxN autoimmune arthritis model, we demonstrated that Peyer's patch (PP) Tfh cells were essential for gut commensal segmented filamentous bacteria (SFB)-induced systemic arthritis despite the production of auto-antibodies predominantly occurring in systemic lymphoid tissues, not PPs. We determined that SFB, by driving differentiation and egress of PP Tfh cells into systemic sites, boosted systemic Tfh cell and auto-antibody responses that exacerbated arthritis. SFB induced PP Tfh cell differentiation by limiting the access of interleukin 2 to CD4(+) T cells, thereby enhancing Tfh cell master regulator Bcl-6 in a dendritic cell-dependent manner. These findings showed that gut microbiota remotely regulated a systemic disease by driving the induction and egress of gut Tfh cells.

Asymmetric Dimethylarginine Stimulates Akt1 Phosphorylation via Heat Shock Protein 70-Facilitated Carboxyl-Terminal Modulator Protein Degradation in Pulmonary Arterial Endothelial Cells.

Asymmetric dimethylarginine (ADMA) induces the mitochondrial translocation of endothelial nitric oxide synthase (eNOS) through the nitration-mediated activation of Akt1. However, it is recognized that the activation of Akt1 requires phosphorylation events at threonine (T) 308 and serine (S) 473. Thus, the current study was performed to elucidate the potential effect of ADMA on Akt1 phosphorylation and the mechanisms that are involved. Exposure of pulmonary arterial endothelial cells to ADMA enhanced Akt1 phosphorylation at both threonine 308 and Ser473 without altering Akt1 protein levels, phosphatase and tensin homolog activity, or membrane Akt1 levels. Heat shock protein (Hsp) 90 plays a pivotal role in maintaining Akt1 activity, and our results demonstrate that ADMA decreased Hsp90-Akt1 interactions, but, surprisingly, overexpression of a dominant-negative Hsp90 mutant increased Akt1 phosphorylation. ADMA exposure or overexpression of dominant-negative Hsp90 increased Hsp70 levels, and depletion of Hsp70 abolished ADMA-induced Akt1 phosphorylation. ADMA decreased the interaction of Akt1 with its endogenous inhibitor, carboxyl-terminal modulator protein (CTMP). This was mediated by the proteasomal-dependent degradation of CTMP. The overexpression of CTMP attenuated ADMA-induced Akt1 phosphorylation at Ser473, eNOS phosphorylation at Ser617, and eNOS mitochondrial translocation. Finally, we found that the mitochondrial translocation of eNOS in our lamb model of pulmonary hypertension is associated with increased Akt1 and eNOS phosphorylation and reduced Akt1-CTMP protein interactions. In conclusion, our data suggest that CTMP is directly involved in ADMA-induced Akt1 phosphorylation in vitro and in vivo, and that increasing CTMP levels may be an avenue to treat pulmonary hypertension.