Search

R4323 --> Impairment of endothelial integrity causes pathological angiogenesis, tissue inflammation or injury, the hallmark of several diseases including acute lung injury (ALI). While it is known that both endothelial cells (EC) and its underlying extracellular matrix (ECM) are required for preserving the function of underlying tissues, much of the efforts to understand the mechanisms regulating these functions have focused mainly on EC. In contrast, ECM has received little attention, even though ECM itself can influence cellular functions by modulating signaling pathways in various cell types. We have shown that the non‐receptor protein tyrosine kinase, focal adhesion kinase (FAK), canonically known to regulate cell attachment to ECM, maintains endothelial integrity. Cells including EC may also release DNA, RNA, and other macromolecules through exosomes to alter paracrine signaling within EC and epithelial barrier. A fundamental unresolved question is whether endothelial‐FAK impacts ECM turnover by EC, and if so, whether this process maintain lung barrier protective signaling through secreting exosomes. Therefore, we have performed mass spectrometry of lungs harvested from control and mice lacking FAK in the endothelium after decellularization. We also isolated exosomes from control and FAK null EC. Our goal here was to determine the role of FAK‐driven ECM and EC‐exosomes in altering the barrier integrity. We found that EC‐FAK deletion markedly altered the proteomic content of lung‐ECM. Deletion of FAK also led to generation of exosomes, which disrupted barrier function of naïve EC. Further experiments are being carried out to characterize the ECM hydrogels and exosomes derived from control or FAK‐null lungs to demonstrate the role of FAK‐regulated ECM in regulating paracrine signaling in maintaining lung barrier integrity. [ABSTRACT FROM AUTHOR]

Increased lung vascular permeability and neutrophilic inflammation are hallmarks of acute lung injury. Alveolar macrophages (AMϕ), the predominant sentinel cell type in the airspace, die in massive numbers while fending off pathogens. Recent studies indicate that the AMϕ pool is replenished by airspace-recruited monocytes, but the mechanisms instructing the conversion of recruited monocytes into reparative AMϕ remain elusive. Cyclic AMP (cAMP) is a vascular barrier protective and immunosuppressive second messenger in the lung. Here, we subjected mice expressing GFP under the control of the Lysozyme-M promoter (LysM-GFP mice) to the LPS model of rapidly resolving lung injury to address the impact of mechanisms determining cAMP levels in AMϕ and regulation of mobilization of the reparative AMϕ-pool. RNA-seq analysis of flow-sorted Mϕ identified phosphodiesterase 4b (PDE4b) as the top LPS-responsive cAMP-regulating gene. We observed that PDE4b expression markedly increased at the time of peak injury (4 h) and then decreased to below the basal level during the resolution phase (24 h). Activation of transcription factor NFATc2 was required for the transcription of PDE4b in Mϕ. Inhibition of PDE4 activity at the time of peak injury, using intratracheal rolipram, increased cAMP levels, augmented the reparative AMϕ pool, and resolved lung injury. This response was not seen following conditional depletion of monocytes, thus establishing airspace-recruited PDE4b-sensitive monocytes as the source of reparative AMϕ. Interestingly, adoptive transfer of rolipram-educated AMϕ into injured mice resolved lung edema. We propose suppression of PDE4b as an effective approach to promote reparative AMϕ generation from monocytes for lung repair.

Acute lung injury (ALI) is a lethal inflammatory lung disorder whose incidence is on the rise. Alveolar macrophages normally act to resolve inflammation, but when dysregulated they can provoke ALI. We demonstrate that monocyte-derived macrophages (CD11b + macrophages) recruited into the airspace upregulate the anti-inflammatory function of alveolar macrophages by suppressing their stimulator of type 1 interferon gene (STING) signaling. Depletion of CD11b + macrophages in mice (macrophage dep mice) after endotoxin or after Pseudomonas aeruginosa causes expansion of the inflammatory alveolar macrophage population, leading to neutrophil accumulation, irreversible loss of lung vascular barrier function, and lethality. We show that CD11b + macrophages suppress alveolar macrophage-STING signaling via sphingosine kinase-2 (SPHK2) generation of sphingosine-1-phosphate (S1P). Thus, adoptive transfer of wild-type (WT) or STING -/- , but not SPHK2 -/- , CD11b monocytes from murine bone marrow into injured macrophage dep mice rescue anti-inflammatory alveolar macrophages and reverse lung vascular injury. SPHK2-induced S1P generation in CD11b + macrophages has the potential to educate alveolar macrophages to resolve ALI., Competing Interests: Declaration of Interests The authors declare no competing interests., (Copyright © 2020 The Author(s). Published by Elsevier Inc. All rights reserved.)

High mobility group A1 (HMGA1), a non-histone chromosomal protein, is highly expressed in a wide range of human cancers including cervical, breast, and prostate cancers. Therefore, hmga1 gene is considered as an attractive potential target for anticancer drugs. We have chosen 27 bp DNA sequence from a regulatory region of hmga1 promoter and studied its interaction with adriamycin (ADM) and in vitro expression of HMGA1 in the presence of ADM in HeLa cell line. A variety of biophysical techniques were employed to understand the characteristics of [DNA-ADM] complex. Spectrophotometric titration data, DNA denaturation profiles, and quenching of fluorescence of ADM in the presence of DNA demonstrated a strong complexation between DNA and ADM with a high binding affinity (Ka) of 1.3 × 10(6) M(-1) and a stoichiometry of 1:3 (drug:nucleotide). The energetics of binding obtained from isothermal titration calorimetry and differential scanning calorimetry suggest the binding to be exothermic and enthalpy (∆H, -6.7 ± 2.4 kcal M(-1)) and entropy (TΔS, 18.5 ± 6.4 kcal M(-1)) driven (20°C), which is typical of intercalative mode of binding. Further, results on decreased expression (by ~70%) of HMGA1 both at mRNA and protein levels in association with the observed cell death (by ~75%) in HeLa cell line, clearly confirm that ADM does target hmga1; however, the effect of ADM on genes other than hmga1 either directly or via hmga1-mediated pathways cannot be ruled out in the observed cytotoxicity. Therefore, hmga1 in general and particularly the regulatory region is a promising target for therapeutic strategy in combating cancer.