Estrogen metabolism in pulmonary arterial hypertension

Pulmonary arterial hypertension (PAH) is a devastating and progressive vasculopathy of the pulmonary arteries for which there is no cure. There is an urgent need for more effective therapies. PAH is characterised by elevated pulmonary arterial pressures and obstructive vascular lesions in the distal vasculature by excessive cellular proliferation. As a result, the right ventricle is placed under excessive strain resulting in adaptive hypertrophy which progresses to maladaptive hypertrophy and failure. PAH is more common in women than in men suggesting that estrogens may be integral to disease pathogenesis. Understanding the biological basis for this sex difference would offer a new treatment paradigm in this devastating cardiovascular disease. Here, we challenged the concept that the estrogen metabolic axis is dysregulated in PAH New insights have revealed a potential contribution of the estrogen metabolizing enzyme, cytochrome P450 1B1 (CYP1B1) in the development of PAH. 17β-estradiol (17β-E2) and estrone (E1) are metabolized by the activity of CYP1B1 to the 2-, 4- and 16-hydroxylated estrogens. Here, we defined the role of CYP1B1 in the pathogenesis of PAH. CYP1B1 expression was increased in both experimental (hypoxia and SU5416+hypoxia) and in heritable and idiopathic PAH (HPAH and IPAH, respectively). Both male and female CYP1B1 knockout mice (CYP1B1-/-) were challenged with chronic hypoxia to induce PAH as assessed by right ventricular systolic pressures (RVSP), right ventricular hypertrophy (RVH) and pulmonary vascular remodeling. CYP1B1-/- mice were protected against hypoxia-induced pulmonary hypertension (PH). CYP1B1 inhibition with the highly potent and selective inhibitor 2,3',4,5'-tetramethoxystilbene (TMS; 3 mg/kg/day by intra-peritoneal injection) attenuated the development of hypoxia-induced PH. Only moderate effects were observed with CYP1B1 inhibition in monocrotaline-induced PH, despite improving survival rates. Female mice that over-express the human serotonin transporter gene (SERT+ mice) develop a spontaneous PAH phenotype at 5 months of age which is dependent on circulating levels of 17β-E2. Here, we provide evidence that the estrogen metabolic axis is dysregulated in these mice and this may underlie their PAH phenotype. The estrogen synthesizing enzyme aromatase and CYP1B1 was increased in whole lung homogenates of female SERT+ mice compared to wild-type mice. Despite increased expression of aromatase, 17β-E2 concentrations were unchanged. CYP1B1 inhibition with TMS (1.5mg/kg/day by intra-peritoneal injection) attenuated the PAH phenotype in female SERT+ mice as assessed by RVSP and pulmonary vascular remodeling Other studies have identified that the 16-hydroxylated metabolites of estrogens (17β-E2 and E1) are the only CYP1B1 metabolites to induce cellular proliferation, with the most profound effects observed with 16α-hydroxyestrone (16α-OHE1). In mice exposed to chronic hypoxia, urinary concentrations of 16α-OHE1 were increased. Chronic dosing of 16α-OHE1 in mice (1.5mg/kg/day by intra-peritoneal injection for 28 days) resulted in the development of a PAH phenotype in female mice only. 16α-OHE1 induced cellular proliferation in human pulmonary arterial smooth muscle cells (hPASMCs) and this was inhibited by a scavenger of reactive oxygen species (ROS) and an inhibitor of extracellular regulated kinase 1/2 (ERK 1/2). 4-hydroxylation is the predominant metabolic pathway activated by CYP1B1 activity and we therefore investigated the effects of the 4-hydroxylated metabolite of 17β-E2 in vivo. 4-hydroxyestradiol (4-OHE2) had no effects on PAH parameters in mice (1.5mg/kg/day by intra-peritoneal injection for 28 days). However, serotonin-induced vasoconstriction of the intra-pulmonary arteries was dramatically reduced in arteries harvested from mice dosed with 4-OHE2. More recent studies have identified that 4-hydroxyestrone (4-OHE1) is the predominant CYP1B1 metabolite in the lungs of mice. Interestingly, despite evidence for a pathogenic function of CYP1B1 activity in vivo, 4-OHE1 inhibited cellular proliferation in hPASMCs as assessed by thymidine incorporation whilst no effects were reported on cell viability. We provide evidence for an altered estrogen metabolic axis in PAH, by in part, overexpression of the putatively pathological CYP1B1. Yet, the dynamic estrogen metabolic profile in pulmonary vascular cells remains undetermined. To address this, we developed a high fidelity HPLC method to quantitatively fate map estrogen metabolism in hPASMCs to determine the dynamic regulation of estrogen metabolism in PAH. We provide the first direct evidence that hPASMCs metabolize 17β-E2 and that estrogen metabolism is pathologically altered in PAH. Our metabolic screen revealed a prominent role for 17β-hydroxysteroid dehydrogenase enzymes in hPASMCs by rapid formation of E1 in all groups studied, increasing with time, with the highest activity in male control hPASMCs and the lowest activity in female control hPASMCs. In female control hPASMCs there was no evidence of CYP activity, whilst numerous metabolites were formed in the other groups studied. The formation of the pathogenic 16α-hydroxylated estrogens was only evident in PASMCs from both male and female PAH patients at 24 and 48 hours. Globally, this study introduces a platform to elucidate effects of PAH insults and potential therapies on the estrogen-metabolic profile in pulmonary vascular cells. Overall, we provide eminent evidence that the estrogen metabolic axis is pathologically altered in PAH and is influenced by gender. This provides a strong rationale for the application of estrogen-sensitive therapies in the management of this highly female discriminating disease

The serotonin transporter promotes a pathological estrogen metabolic pathway in pulmonary hypertension via cytochrome P450 1B1 pulmonary circulation

Pulmonary arterial hypertension (PAH) is a devastating vasculopathy that predominates in women and has been associated with dysregulated estrogen and serotonin signaling. Overexpression of the serotonin transporter (SERT+) in mice results in an estrogen-dependent development of pulmonary hypertension (PH). Estrogen metabolism by cytochrome P450 1B1 (CYP1B1) contributes to the pathogenesis of PAH, and serotonin can increase CYP1B1 expression in human pulmonary arterial smooth muscle cells (hPASMCs). We hypothesized that an increase in intracellular serotonin via increased SERT expression may dysregulate estrogen metabolism via CYP1B1 to facilitate PAH. Consistent with this hypothesis, we found elevated lung CYP1B1 protein expression in female SERT+ mice accompanied by PH, which was attenuated by the CYP1B1 inhibitor 2,3',4,5'-tetramethoxystilbene (TMS). Lungs from female SERT+ mice demonstrated an increase in oxidative stress that was marked by the expression of 8-hydroxyguanosine; however, this was unaffected by CYP1B1 inhibition. SERT expression was increased in monocrotaline-induced PH in female rats; however, TMS did not reverse PH in monocrotaline-treated rats but prolonged survival. Stimulation of hPASMCs with the CYP1B1 metabolite 16α-hydroxyestrone increased cellular proliferation, which was attenuated by an inhibitor (MPP) of estrogen receptor alpha (ERα) and a specific ERα antibody. Thus, increased intracellular serotonin caused by increased SERT expression may contribute to PAH pathobiology by dysregulation of estrogen metabolic pathways via increased CYP1B1 activity. This promotes PASMC proliferation by the formation of pathogenic metabolites of estrogen that mediate their effects via ERα. Our studies indicate that targeting this pathway in PAH may provide a promising antiproliferative therapeutic strategy

TRIM27 is an autophagy substrate facilitating mitochondria clustering and mitophagy via phosphorylated TBK1

Tripartite motif-containing protein 27 (TRIM27/also called RFP) is a multifunctional ubiquitin E3 ligase involved in numerous cellular functions, such as proliferation, apoptosis, regulation of the NF-kB pathway, endosomal recycling and the innate immune response. TRIM27 interacts directly with TANK-binding kinase 1 (TBK1) and regulates its stability. TBK1 in complex with autophagy receptors is recruited to ubiquitin chains assembled on the mitochondrial outer membrane promoting mitophagy. Here, we identify TRIM27 as an autophagy substrate, depending on ATG7, ATG9 and autophagy receptors for its lysosomal degradation. We show that TRIM27 forms ubiquitylated cytoplasmic bodies that co-localize with autophagy receptors. Surprisingly, we observed that induced expression of EGFP-TRIM27 in HEK293 FlpIn TRIM27 knockout cells mediates mitochondrial clustering. TRIM27 interacts with autophagy receptor SQSTM1/p62, and the TRIM27-mediated mitochondrial clustering is facilitated by SQSTM/p62. We show that phosphorylated TBK1 is recruited to the clustered mitochondria. Moreover, induced mitophagy activity is reduced in HEK293 FlpIn TRIM27 knockout cells, while re-introduction of EGFP-TRIM27 completely restores the mitophagy activity. Inhibition of TBK1 reduces mitophagy in HEK293 FlpIn cells and in the reconstituted EGFP-TRIM27-expressing cells, but not in HEK293 FlpIn TRIM27 knockout cells. Altogether, these data reveal novel roles for TRIM27 in mitophagy, facilitating mitochondrial clustering via SQSTM1/p62 and mitophagy via stabilization of phosphorylated TBK1 on mitochondria

Omicron Variant Generates a Higher and More Sustained Viral Load in Nasopharynx and Saliva Than the Delta Variant of SARS-CoV-2

The Omicron variant of SARS-CoV-2 spreads more easily than earlier variants, possibly as a result of a higher viral load in the upper respiratory tract and oral cavity. Hence, we investigated whether the Omicron variant generates a higher viral load than that of the Delta variant in saliva and nasopharynx. Both specimens were collected from 52 Omicron and 17 Delta cases at two time points one week apart and analyzed by qRT-PCR. Viral load was measured as 10 log RNA genome copies per 1000 human cells according to the WHO reference standard. We found that Omicron cases carried a higher viral load and had more sustained viral shedding compared to the Delta cases, especially in the nasopharynx

Thymosin β4 and prothymosin α promote cardiac regeneration post-ischaemic injury in mice

Aims The adult mammalian heart is a post-mitotic organ. Even in response to necrotic injuries, where regeneration would be essential to reinstate cardiac structure and function, only a minor percentage of cardiomyocytes undergo cytokinesis. The gene programme that promotes cell division within this population of cardiomyocytes is not fully understood. In this study, we aimed to determine the gene expression profile of proliferating adult cardiomyocytes in the mammalian heart after myocardial ischaemia, to identify factors to can promote cardiac regeneration. Methods and results Here, we demonstrate increased 5-ethynyl-2’deoxyuridine incorporation in cardiomyocytes 3 days post-myocardial infarction in mice. By applying multi-colour lineage tracing, we show that this is paralleled by clonal expansion of cardiomyocytes in the border-zone of the infarcted tissue. Bioinformatic analysis of single-cell RNA sequencing data from cardiomyocytes at 3 days post ischaemic injury revealed a distinct transcriptional profile in cardiomyocytes expressing cell cycle markers. Combinatorial overexpression of the enriched genes within this population in neonatal rat cardiomyocytes and mice at postnatal day 12 (P12) unveiled key genes that promoted increased cardiomyocyte proliferation. Therapeutic delivery of these gene cocktails into the myocardial wall after ischaemic injury demonstrated that a combination of thymosin beta 4 (TMSB4) and prothymosin alpha (PTMA) provide a permissive environment for cardiomyocyte proliferation and thereby attenuated cardiac dysfunction. Conclusion This study reveals the transcriptional profile of proliferating cardiomyocytes in the ischaemic heart and shows that overexpression of the two identified factors, TMSB4 and PTMA, can promote cardiac regeneration. This work indicates that in addition to activating cardiomyocyte proliferation, a supportive environment is a key for regeneration to occur

Rpl3l gene deletion in mice reduces heart weight over time

Introduction: The ribosomal protein L3-like (RPL3L) is a heart and skeletal muscle-specific ribosomal protein and paralogue of the more ubiquitously expressed RPL3 protein. Mutations in the human RPL3L gene are linked to childhood cardiomyopathy and age-related atrial fibrillation, yet the function of RPL3L in the mammalian heart remains unknown.Methods and Results: Here, we observed that mouse cardiac ventricles express RPL3 at birth, where it is gradually replaced by RPL3L in adulthood but re-expressed with induction of hypertrophy in adults. Rpl3l gene-deleted mice were generated to examine the role of this gene in the heart, although Rpl3l−/− mice showed no overt changes in cardiac structure or function at baseline or after pressure overload hypertrophy, likely because RPL3 expression was upregulated and maintained in adulthood. mRNA expression analysis and ribosome profiling failed to show differences between the hearts of Rpl3l null and wild type mice in adulthood. Moreover, ribosomes lacking RPL3L showed no differences in localization within cardiomyocytes compared to wild type controls, nor was there an alteration in cardiac tissue ultrastructure or mitochondrial function in adult Rpl3l−/− mice. Similarly, overexpression of either RPL3 or RPL3L with adeno-associated virus −9 in the hearts of mice did not cause discernable pathology. However, by 18 months of age Rpl3l−/− null mice had significantly smaller hearts compared to wild type littermates.Conclusion: Thus, deletion of Rpl3l forces maintenance of RPL3 expression within the heart that appears to fully compensate for the loss of RPL3L, although older Rpl3l−/− mice showed a mild but significant reduction in heart weight

Breakthrough infections with the omicron and delta variants of SARS-CoV-2 result in similar re-activation of vaccine-induced immunity

Background: Results showing that sera from double vaccinated individuals have minimal neutralizing activity against Omicron have been interpreted as indicating the need for a third vaccine dose for protection. However, there is little information about early immune responses to Omicron infection in double vaccinated individuals. Methods: We measured inflammatory mediators, antibodies to the SARS-CoV-2 spike and nucleocapsid proteins, and spike peptide-induced release of interferon gamma in whole blood in 51 double-vaccinated individuals infected with Omicron, in 14 infected with Delta, and in 18 healthy controls. The median time points for the first and second samples were 7 and 14 days after symptom onset, respectively. Findings: Infection with Omicron or Delta led to a rapid and similar increase in antibodies to the receptor-binding domain (RBD) of Omicron protein and spike peptide-induced interferon gamma in whole blood. Both the Omicron- and the Delta-infected patients had a mild and transient increase in inflammatory parameters. <p<Interpretation: The results suggest that two vaccine doses are sufficient to mount a rapid and potent immune response upon infection in healthy individuals of with the Omicron variant