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Pulmonary arterial hypertension (PAH) is a devastating and progressive disease with limited treatment options. Endothelial dysfunction plays a central role in development and progression of PAH, yet the underlying mechanisms are incompletely understood. The endosome-lysosome system is important to maintain cellular health and the small GTPase RAB7 regulates many functions of this system. Here, we explored the role of RAB7 in endothelial cell (EC) function and lung vascular homeostasis. We found reduced expression of RAB7 in ECs from PAH patients. Endothelial haploinsufficiency of RAB7 caused spontaneous PH in mice. Silencing of RAB7 in ECs induced broad changes in gene expression revealed via RNA sequencing and RAB7 silenced ECs showed impaired angiogenesis, expansion of a senescent cell fraction, combined with impaired endolysosomal trafficking and degradation, which suggests inhibition of autophagy at the pre-degradation level. Further, mitochondrial membrane potential and oxidative phosphorylation were decreased, and glycolysis was enhanced. Treatment with the RAB7 activator ML-098 reduced established PH in chronic hypoxia/SU5416 rats. In conclusion, we demonstrate here for the first time the fundamental impairment of EC function by loss of RAB7 that leads to PH and show RAB7 activation as a potential therapeutic strategy in a preclinical model of PH.

Cardiovascular sequelae of severe acute respiratory syndrome (SARS) coronavirus-2 (CoV-2) disease 2019 (COVID-19) contribute to the complications of the disease. One potential complication is lung vascular remodeling, but the exact cause is still unknown. We hypothesized that endothelial TLR3 insufficiency contributes to lung vascular remodeling induced by SARS-CoV-2. In the lungs of COVID-19 patients and SARS-CoV-2 infected Syrian hamsters, we discovered thickening of the pulmonary artery media and microvascular rarefaction, which were associated with decreased TLR3 expression in lung tissue and pulmonary artery endothelial cells (ECs).In vitro, SARS-CoV-2 infection reduced endothelial TLR3 expression. Following infection with mouse-adapted (MA) SARS-CoV-2, TLR3 knockout mice displayed heightened pulmonary artery remodeling and endothelial apoptosis. Treatment with the TLR3 agonist polyinosinic:polycytidylic acid reduced lung tissue damage, lung vascular remodeling, and endothelial apoptosis associated with MA SARS-CoV-2 infection. In conclusion, repression of endothelial TLR3 is a potential mechanism of SARS-CoV-2 infection associated lung vascular remodeling and enhancing TLR3 signaling is a potential strategy for treatment.

Pulmonary arterial hypertension (PAH) is a progressive, devastating disease, and its main histological manifestation is an occlusive pulmonary arteriopathy. One important functional component of PAH is aberrant endothelial cell (EC) function including apoptosis-resistance, unchecked proliferation, and impaired migration. The mechanisms leading to and maintaining physiologic and aberrant EC function are not fully understood. Here, we tested the hypothesis that in PAH, ECs have increased expression of the transmembrane protein integrin-β5, which contributes to migration and survival under physiologic and pathological conditions, but also to endothelial-to-mesenchymal transition (EnMT). We found that elevated integrin-β5 expression in pulmonary artery lesions and lung tissue from PAH patients and rats with PH induced by chronic hypoxia and injection of CD117

Cysteine3Histidine (CCCH)-type zinc finger proteins comprise a large family that is well conserved across eukaryotes. Among them, tandem CCCH zinc finger proteins (TZFs) play critical roles in mRNA metabolism in animals and yeast. While there are only three TZF members in humans, a much higher number of TZFs has been found in many plant species. Notably, plant TZFs are over-represented by a class of proteins containing a unique TZF domain preceded by an arginine (R)-rich (RR) motif, hereafter called RR-TZF. Recently, there have been a large number of reports indicating that RR-TZF proteins can localize to processing bodies (P-bodies) and stress granules (SG), two novel cytoplasmic aggregations of messenger ribonucleoprotein complexes (mRNPs), and play critical roles in plant growth, development and stress response, probably via RNA regulation. This review focuses on the classification and most recent development of molecular, cellular and genetic analyses of plant RR-TZF proteins.

Tandem CCCH zinc finger proteins (TZFs) are post-transcriptional regulators of gene expression in animals and yeast. Genetic studies indicate that plant TZFs are involved in hormone-mediated developmental and environmental responses. We have demonstrated previously that Arabidopsis AtTZF1 can localize to processing bodies (PBs) and stress granules (SGs), and affects abscisic acid (ABA)- and gibberellic acid (GA)-mediated growth, stress and gene expression responses. Here we show that AtTZF4, 5 and 6 are specifically expressed in seeds. Consistent with the observation that their expression levels decline during seed imbibition, AtTZF4, 5 and 6 are up-regulated by ABA and down-regulated by GA. Mutant analyses indicate that AtTZF4, 5 and 6 act as positive regulators for ABA- and negative regulators for light- and GA-mediated seed germination responses. Results of gene expression analysis indicate that AtTZF4, 5 and 6 affect seed germination by controlling genes critical for ABA and GA response. Furthermore, AtTZF4, 5 and 6 can co-localize with both PB and SG markers in Arabidopsis cells. Specifically, AtTZF6 can be assembled into PBs and SGs in embryos with the induction of stress hormone methyl jasmonate under the control of native AtTZF6 promoter.

Although multiple lines of evidence have indicated that Arabidopsis thaliana Tandem CCCH Zinc Finger proteins, AtTZF4, 5 and 6 are involved in ABA, GA and phytochrome mediated seed germination responses, the interacting proteins involved in these processes are unknown. Using yeast two-hybrid screens, we have identified 35 putative AtTZF5 interacting protein partners. Among them, Mediator of ABA-Regulated Dormancy 1 (MARD1) is highly expressed in seeds and involved in ABA signal transduction, while Responsive to Dehydration 21A (RD21A) is a well-documented stress responsive protein. Co-immunoprecipitation (Co-IP) and bimolecular fluorescence complementation (BiFC) assays were used to confirm that AtTZF5 can interact with MARD1 and RD21A in plant cells, and the interaction is mediated through TZF motif. In addition, AtTZF4 and 6 could also interact with MARD1 and RD21A in Y-2-H and BiFC assay, respectively. The protein-protein interactions apparently take place in processing bodies (PBs) and stress granules (SGs), because AtTZF5, MARD1 and RD21A could interact and co-localize with each other and they all can co-localize with the same PB and SG markers in plant cells.

Tandem CCCH zinc finger proteins (TZFs) are post-transcriptional regulators of gene expression in animals and yeast. Genetic studies indicate that plant TZFs are involved in hormone-mediated developmental and environmental responses. We have demonstrated previously that Arabidopsis AtTZF1 can localize to processing bodies (PBs) and stress granules (SGs), and affects abscisic acid (ABA)- and gibberellic acid (GA)-mediated growth, stress and gene expression responses. Here we show that AtTZF4, 5 and 6 are specifically expressed in seeds. Consistent with the observation that their expression levels decline during seed imbibition, AtTZF4, 5 and 6 are up-regulated by ABA and down-regulated by GA. Mutant analyses indicate that AtTZF4, 5 and 6 act as positive regulators for ABA- and negative regulators for light- and GA-mediated seed germination responses. Results of gene expression analysis indicate that AtTZF4, 5 and 6 affect seed germination by controlling genes critical for ABA and GA response. Furthermore, AtTZF4, 5 and 6 can co-localize with both PB and SG markers in Arabidopsis cells. Specifically, AtTZF6 can be assembled into PBs and SGs in embryos with the induction of stress hormone methyl jasmonate under the control of native AtTZF6 promoter.