Protection of endothelial integrity has been recognized as a frontline approach to alleviating sepsis progression, yet no effective agent for preserving endothelial integrity is available. Using an unusual anti-angiopoietin 2 (ANG2) antibody, ABTAA (ANG2-binding and TIE2-activating antibody), we show that activation of the endothelial receptor TIE2 protects the vasculature from septic damage and provides survival benefit in three sepsis mouse models. Upon binding to ANG2, ABTAA triggers clustering of ANG2, assembling an ABTAA/ANG2 complex that can subsequently bind and activate TIE2. Compared with a conventional ANG2-blocking antibody, ABTAA was highly effective in augmenting survival from sepsis by strengthening the endothelial glycocalyx, reducing cytokine storms, vascular leakage, and rarefaction, and mitigating organ damage. Together, our data advance the role of TIE2 activation in ameliorating sepsis progression and open a potential therapeutic avenue for sepsis to address the lack of sepsis-specific treatment.

The lung is highly vulnerable during sepsis, yet its functional deterioration accompanied by disturbances in the pulmonary microcirculation is poorly understood. This study aimed to investigate how the pulmonary microcirculation is distorted in sepsis-induced acute lung injury (ALI) and reveal the underlying cellular pathophysiologic mechanism.Using a custom-made intravital lung microscopic imaging system in a murine model of sepsis-induced ALI, we achieved direct real-time visualisation of the pulmonary microcirculation and circulating cellsin vivo. We derived the functional capillary ratio (FCR) as a quantitative parameter for assessing the fraction of functional microvasculature in the pulmonary microcirculation and dead space.We identified that the FCR rapidly decreases in the early stage of sepsis-induced ALI. The intravital imaging revealed that this decrease resulted from the generation of dead space, which was induced by prolonged neutrophil entrapment within the capillaries. We further showed that the neutrophils had an extended sequestration time and an arrest-like dynamic behaviour, both of which triggered neutrophil aggregates inside the capillaries and arterioles. Finally, we found that Mac-1 (CD11b/CD18) was upregulated in the sequestered neutrophils and that a Mac-1 inhibitor restored the FCR and improved hypoxaemia.Using the intravital lung imaging system, we observed that Mac-1-upregulated neutrophil aggregates led to the generation of dead space in the pulmonary microcirculation that was recovered by a Mac-1 inhibitor in sepsis-induced ALI.

In the central nervous system, endothelial cells (ECs) and pericytes (PCs) of blood vessel walls cooperatively form a physical and chemical barrier to maintain neural homeostasis. However, in diabetic retinopathy (DR), the loss of PCs from vessel walls is assumed to cause breakdown of the blood-retina barrier (BRB) and subsequent vision-threatening vascular dysfunctions. Nonetheless, the lack of adequate DR animal models has precluded disease understanding and drug discovery. Here, by using an anti-PDGFRβ antibody, we show that transient inhibition of the PC recruitment to developing retinal vessels sustained EC-PC dissociations and BRB breakdown in adult mouse retinas, reproducing characteristic features of DR such as hyperpermeability, hypoperfusion, and neoangiogenesis. Notably, PC depletion directly induced inflammatory responses in ECs and perivascular infiltration of macrophages, whereby macrophage-derived VEGF and placental growth factor (PlGF) activated VEGFR1 in macrophages and VEGFR2 in ECs. Moreover, angiopoietin-2 (Angpt2) upregulation and Tie1 downregulation activated FOXO1 in PC-free ECs locally at the leaky aneurysms. This cycle of vessel damage was shut down by simultaneously blocking VEGF, PlGF, and Angpt2, thus restoring the BRB integrity. Together, our model provides new opportunities for identifying the sequential events triggered by PC deficiency, not only in DR, but also in various neurological disorders.

When the rnpB gene encoding M1 RNA, the RNA component of Escherichia coli RNase P, is transcribed, the primary M1 RNA transcript (pM1 RNA) is produced and subsequently processed at the 3 end to generate the mature M1 RNA. To study features of pM1 RNA thought to be involved in RNA processing, systematic mutations were introduced in sequence elements and secondary structures surrounding the processing site using p23 RNA, a truncated pM1 RNA transcribed from the internally deleted rnpB gene, as a model substrate and the processing of its mutant derivatives was analyzed in vivo and in vitro. Neither the alteration of two bases forming the processing site nor the disruption of secondary structures surrounding the site significantly affected the processing efficiency although the secondary structures were required for maintaining RNA stability. In contrast, mutations at the rne-dependent site, GAUUU, immediately 3 to the processing site inhibited the processing and the extent of the inhibition varied with the altered sequences. Furthermore, the processing of the mutants of the rne-dependent site as well as wild-type p23 RNA was inhibited in an E. coli rne ts strain at the nonpermissive temperature.Ribonuclease P (RNase P) is a processing enzyme that catalyzes the endonucleolytic removal of 5Ј leader sequences from precursors of tRNAs to generate the mature 5Ј ends of tRNAs (1). In Escherichia coli, RNase P is composed of the RNA subunit (M1 RNA, 377 nucleotides) and the protein subunit (C5 protein, 119 amino acid residues) (2-4).The promoter region of the rnpB gene coding for M1 RNA contains three regions of homology to the E. coli consensus promoter sequence (5). In vitro and in vivo transcriptions arise from the nearest promoter, P-1, almost exclusively (6), even though transcripts can originate from the upstream promoters (5). The 3Ј-flanking region of the rnpB gene contains a repeated DNA sequence of 113 base pairs. This sequence, which includes both the 3Ј terminal 24 nucleotides of the M1 RNA coding sequence and the region for transcription termination, is repeated almost 3.5 times (7). The region for transcription termination contains a stem and loop structure characteristic of structures found near regions of -independent transcription termination (8), and functions in a -independent fashion in vitro (9). In vivo studies using the galactokinase transcription fusion plasmids (10) indicate that more than 94% of transcription is terminated at the first terminator, T1, in the first repeating unit. Therefore, the RNA product initiated from P-1 and terminated at T1 should be a major primary transcript of the rnpB gene from which mature M1 RNA is derived. This transcript, termed pM1 RNA (primary transcript), carries extra stretches of 36 nucleotides containing a termination stem and loop at the 3Ј end of M1 RNA which are removed by a processing event. pM1 RNA of 413 nucleotides is processed to form mature M1 RNA in vitro by an S30 extract (9). On the other hand, a 40% ammonium sulfate precipitate of S200 extract from...

No abstract