A major clinical hallmark of SFG rickettsial disease is the infection of ECs leading to enhanced vascular permeability . In the present study using a HUVEC model, we confirmed previous observations from different in vitro endothelial cell models [49–51] that endothelial permeability was enhanced upon infection endothelial cells with SFG R. conorii. However, the cellular and molecular mechanisms by which SFG rickettsiae increase endothelial permeability remain to be elucidated. Our previous investigation  demonstrated that tyrosine phosphorylation of endothelial adherens junctional protein VE-cadherins involved alterations in calcium-dependent homophilic protein-protein adhesion forces between VE-cadherins on adjacent cells, underlying endothelial paracellular dysfunction following SFG rickettsial infection. Therefore, phosphorylation of transmembrane adhesion proteins may serve as a pivotal regulator in endothelial barrier dysfunction. Yet the implied direct association and the pathway to link SFG rickettsial infection of the endothelium and VE-cadherin phosphorylation remain unproven.
There are many mechanisms that regulate VE-cadherin stability at adherens junctions through modulating phosphorylation that effectively control VE-cadherin availability at the endothelial surface [10, 15]. Angiogenic stimuli such as vascular endothelial growth factor (VEGF) , epidermal growth factor (EGF) , fibroblast growth factor (FGF) , and angiopoietin-1  have produced inconsistent effects on induction of tyrosine phosphorylation of VE-cadherin. The literature suggests that VEGF and EGF may trigger tyrosine phosphorylation of VE-cadherin, disassembling the adherens junction complex through Src and Rac pathways [14, 15]; angiopoietin-1 and FGF may dephosphorylate VE-cadherin to stabilize the endothelial barrier apparatus associated with β-integrin [12, 16]. Observations from the present study displayed that ANG, an early identified angiogenic factor, was remarkably increased in endothelial layers in the microvasculature in multiple organs post-infection with R. conorii, and co-localized with SFG rickettsiae in the lesion. In liver, greater accumulation of ANG located at the endothelial layer of hepatic sinusoidal blood vessels compared to hepatocytes (Figure 2H) suggest hepatocytes as potential sources of increased synthesis of ANG after SFG rickettsial infection, but endothelial cells are the targets of upregulated ANG.
ANG was originally isolated from conditioned media of human colon adenocarcinoma cells based on its angiogenic activity . To induce a wide range of cellular responses, ANG must undergo nuclear translocation and enhance rRNA transcription, the rate-limiting step of ribosome biogenesis and cell growth [21, 53]. However, in vitro studies showed that confluent quiescent endothelial cells do not internalize exogenous ANG . In the present study, rickettsial infection sensitized confluent HUVECs and initiated translocation of exogenous ANG in a compartmentalized pattern at different times post-infection (Figure 3). Typically, at 24 hrs post-infection, exogenous ANGs were in the nuclei. In contrast, at 72 hrs p.i. translocation of ANG predominated in cytoplasm. The underlying mechanisms associated with compartmentalized translocation during different intervals post-infection by SFG rickettsiae still needs to be investigated. Current evidence suggests that nuclear translocation of ANG is via an unidentified receptor(s) system and independent of lysosomes and the microtubule system [18, 25], but is strictly dependent on endothelial cell density . These conditions imply the probability that suppressive signaling from confluent quiescent endothelial cell–cell adhesion blocks endocytosis of ANG, or activation of an unknown ANG-specific transporting system by adverse environments (e.g., stress or intracellular pathogen invasion) initiates internalization of ANG. These two may function as an off–on switch.
Recent studies on the biological activity of ANG have been extended from enabling cell growth and proliferation to sustaining endothelial cell survival under adverse conditions [26–29]. Such emerging roles of ANG are distinct from those of nuclear located ANG. Nuclear translocation in growing endothelial cells and malignant cells correlates with cell proliferation through promoting rRNA transcription and protein synthesis [20, 21]. The role of ANG under stress is envisioned to be activated in the cytoplasm and to mediate reprogramming of global protein translation, saving anabolic energy, and promoting cell survival during adverse events . Our data directly demonstrated that SFG rickettsial infection of endothelial cells induced dynamic translocation of exogenous ANG into different endothelial compartments at different times post-infection. During early infection, nuclear translocation of ANG was not associated with functional evidence of increased endothelial permeability. The illustrated translocation of ANG in cytoplasmic compartments at 72 hrs p.i. (Figure 3) was concomitant with increased endothelial permeability, which was moderated by an ANG translocation inhibitor (Figure 4). Given the facts that phosphorylation of VE-cadherin was enhanced and endocytosis of disassembled VE-cadherin increased when cytoplasmic translocation of exogenous ANG took place, we conclude that, in addition to its angiogenic role, ANG is a key enhancing factor on endothelial barrier dysfunction. The mechanism by which SFG rickettsial infection-triggered cytoplasmic translocation of ANG results in phosphorylation of VE-cadherin mediated endothelial barrier dysfunction has yet to be determined.
ANG is known as the only angiogenic protein to specifically cleave tRNA in vivo and in vitro[18, 42]. Although ANG protein consists of 123 amino acid residues , three distinct functional sites have been identified, including a receptor-binding site, a nuclear localization sequence, and a catalytic site executing ribonucleolytic activity [53, 55, 56]. Our in vitro assay demonstrated that ANG indeed cleaved human tRNAs to produce tRFs. Also, our studies of mouse tissues and HUVECs showed a clear correlation of cellular tRF-5 generation with the cytoplasmic localization of ANG, suggesting that tRNA cleavage by ANG also occurs in vivo when endothelial cells are infected with SFG rickettsiae. Our data showed that the major cleavage products were tRF-5s longer than 30 nts, and these tRF-5s were similar in length to the previously identified tRNA halves produced by a specific cleavage at the anti-codon loop .
In addition, we detected smaller tRF-5s of ~23 nts (for example, tRF5-ValGTG(s) in Figure 1C). These have also been described in human cancer cell lines [36, 37]. One report showed that these are generated by Dicer, however, we do not know if this is also the case in the context of SFG rickettsial infection of endothelial cells. We examined microRNA expression profiles in our deep sequencing data, and failed to detect any global changes upon rickettsial infection, indicating that Dicer activity in general did not change. Also, it is not clear whether tRF5-ValGTG(s) is generated by ANG. In any case, these small tRF-5s were minor in quantity, relative to the longer tRFs, which we therefore pursued as a higher priority.
Our next study aim will be functional evaluation in the HUVEC model. We hypothesize that tRF-5s play a regulatory role by recognizing their target mRNAs in base-pairing interactions. This concept has been supported by recent experimental evidence . We attempted to identify a target mRNA with functional significance. As shown in Table 3, our computational predictions indicated that tRF5-ValGTG and tRF5-GlyGCCtRFs have the potential to interact with transcripts associated with endothelial barrier function, host cell inflammatory response, and autophagy. These are direct topics to pursue in our future studies.
There were a number of limitations in our present study. First, use of neomycin could moderate the ability of exogenous ANGs to enhance endothelial permeability, yet there was elevated endothelial permeability compared to SFG rickettsial infection alone (Figure 4). To address this, future studies can include search for a specific noncellular-toxic ANG receptor inhibitor, other yet to be indentified pathways of tRFs biogenesis, and potential crosstalk between ANG-tRNA-derived sncRNAs and other pathways.
Second, regarding tRF profiling, although several tRFs were identified in the HUVEC model of SFG rickettsial infection and will be the subject of our next functional studies on HUVECs, we do not know the differences between primary human endothelial cells and endothelium in the mouse model. While the core components of the vertebrate microRNA pathway are highly conserved among species, the overall scenario of tRF is not well-understood. For long-term aims to study the functional role of tRFs in primary human endothelial cells, deep-sequence analysis and characterization of sncRNA in the HUVEC model is warranted.
Third and most importantly, the mechanism that regulates switching between the two distinct functional roles of ANG, promoting rRNA transcription in the nucleus and cleaving tRNA in the cytoplasm, is largely unexplored. The survival of mammalian cells exposed to stress requires a reprogramming of protein translation, which is regulated by a family of eukaryotic initiation factors (elF) . Transfection of stress-induced tRFs could repress translation of mRNAs encoding “housekeeping” proteins and trigger the phospho-elF2α-independent stress granules that are essential components of the stress response [27, 29]. Displacement of the elF4F complex is one mechanism by which stress-induced tRFs reprogram protein synthesis . Data from the present study indicate that SFG rickettsial infection of endothelial cells could induce tRNA derived tRFs, and may protect endothelial cells post-infection. However, subsequently triggered endothelial barrier dysfunction causes secondary injury in tissues and organs, including edema and hypoxia. The underlying mechanisms that regulate this biopathologic outcome are largely unknown and require further study.
Analysis of the interactions among enriched tRFs and potential mRNA targets has provided putative mRNA candidates for future studies. There were three tRF-mRNA interactions that attracted our immediate and high priority attention for future studies. The first is the tRF5 interaction with mRNA encoding ValGTC-β protein kinase C (PRKCB). PRKCB has been reported to be involved in several different cellular functions, such as endothelial cell proliferation and barrier function, B-cell activation, and induction of apoptosis [47, 57, 58]. Potential inhibition of this mRNA by this tRF is quite possibly relevant to the pathogenic mechanism in SFG rickettsioses. The second is tRF5-GlyGCC interaction with mRNA that encodes for syntrophin. Syntrophins are adapter proteins that use multiple protein interaction domains to localize a variety of signaling proteins to specific intracellular locations. These include nitric oxide synthase (iNOS and eNOS) that is important for intracellular killing, kinases, ion channels, and water channels [46, 59, 60]. This tRF5-mRNA interaction is also a putative topic for further functional studies in regard to immunity to and pathogenesis of SFG rickettsiae. tRF5-GlyGCC is the third interaction with mRNA, and we identified a target which encodes for endophilin B1 ( also known as Bax-interactin factor 1). Endophilin B1 has been reported to be a key regulator in autophagy through the Bcl2-associated X (Bax) protein pathway [48, 61, 62]. Further research into the roles of tRFs could potentially increase our knowledge regarding the pathogenesis of SFG rickettsiae.