Environmental disruption alters circadian clock gene expression, increasing the risk of adverse cardiac events and suggesting cardiomyocyte-specific circadian responses to external stimuli. Analyses of previously reported transcriptomic data revealed increased expression of Titin-cap (Tcap) in adult compared to embryonic myocytes and identified myosin light chain 2 (Myl2) as clock-controlled. Given cardiac sarcomeric roles of the encoded proteins, we hypothesized that extracellular cues driving postnatal cardiac maturation and hypertrophy influence time-of-day Tcap and Myl2 expression. Tcap induction was concomitant with neonatal myocyte binucleation, fetal gene suppression, and increased heart weight during the early phase of cardiac growth. Since norepinephrine stimulates β-adrenergic and α-adrenergic receptors, the latter driving clock-controlled transcriptional remodeling, phase‑response curves of the β‑adrenergic agonist isoproterenol (ISO) following α-adrenergic stimulation with phenylephrine (PE) were performed on neonatal rat ventricular myocytes (NRVM), revealing periodic myocyte hypertrophy and TCAP protein expression. PE entrenched ISO-mediated Tcap suppression, to which oscillatory Per2 and Myl2 transcription was impervious. Differential NRVM culture density revealed biomass-dependent changes in Per2 and Tcap transcription and hypertrophy timing. Hypoxia initiated myocyte atrophy and Bmal1-dependent Tcap transcription, reflected in decreased heart weight and increased Tcap expression in hypoxic neonatal rat hearts. Tcap depletion impaired Bmal1 and fetal hypertrophic gene expression, compromised hypoxia-mediated Myl2 transcriptional suppression, and aggravated hypoxia-induced atrophy. In summary, Tcap and Myl2 are circadian genes differentially influenced by environmental factors, including adrenergic stimulation, paracrine signaling, and O2 tension during postnatal cardiac maturation. These findings have implications for the distinct regulation of myocyte growth and maturation, by external cues during the postnatal period.

BACKGROUND:Pulmonary arterial hypertension (PAH) presents as increased pressure in the pulmonary arteries (PA) leading to cardiac right ventricular (RV) failure and death. Pulmonary arterial (PA) remodeling characterized by enhanced proliferation of pulmonary arterial smooth muscle cells (PASMC) and fibroblasts (PAFB) underlies PAH. There are currently no cures and PAH mortality remains high. PAH has a striking female-predominant incidence - 4:1 ratio - indicating that males may have a protective factor. However, to date only a few sex-biased factors in PAH have been investigated. METHODS:Analyses were performed on a publicly available microarray dataset (GSE117261), comprising human lung tissues from PAH patients and healthy controls, as well as publicly available single-cell lung atlases of humans and mice. Lung tissue, plasma, PASMC and PAFB were collected from male and female PAH patients. Cell proliferation was assessed after recombinant HGF protein stimulation. PH was induced in male and female rats by monocrotaline (MCT). Lung-specific knockdown was performed by intratracheal siRNA instillation the first two weeks after MCT injection. PA and RV function were assessed by echocardiography, RV systolic pressure by catheterization, and PA remodeling by histology. RESULTS:HGF was only upregulated in lungs of male PAH patients compared to male control lungs, but not in female PAH patients vs. female controls. Elevated plasma HGF correlated with favorable clinical characteristics only in male PAH patients. HGF is highly expressed in vascular SMC and FB in the lung and recombinant HGF inhibited PASMC and PAFB proliferation to a greater extent in cells isolated from male PAH patients compared to female. Lung HGF expression is increased to a higher extent and longer duration at early stage of PH in male rats in MCT model vs. female rats. Finally, knockdown of HGF in the lungs in early disease stage exacerbated PH in male rats characterized by higher mortality, worsened RV and PA function as well as enhanced PA medial thickening and adventitial fibrosis. CONCLUSIONS:Lung HGF expression may be upregulated to counteract PAH disease progression by inhibiting proliferation of PASMC and PAFB. Elevated HGF in males might at least partially account for the lower incidence of male PAH patients.

Background: Pulmonary diseases have sex-specific predilections across the lifespan. The rigor of preclinical research is paramount to ensure the reproducibility and applicability of findings to clinical studies. The overarching goal was to identify current research gaps and the need for consideration of sex as a biological variable (SABV) in preclinical pulmonary research. The objective was to provide a roadmap and the best standards to incorporate and investigate the role of biological sex in preclinical models of lung diseases. Methods: A multidisciplinary working group of 17 international investigators from the American Thoracic Society Assembly on Allergy, Immunology, and Inflammation, external content experts, and researchers engaged in lung basic and translational research. They reviewed the literature, identified critical knowledge gaps, and provided recommendations. Results: The research statement provides an updated summary of the currently available evidence on the standards of SABV research in preclinical models and then offers specific research recommendations focused on the needs of researchers in the pulmonary field. The statement identifies knowledge gaps and develops guidance for experimental design and key considerations for incorporating SABV in two major topic areas: 1) in vivo; and 2) in vitro models. Furthermore, the group developed a checklist to guide researchers in including SABV in preclinical studies. Conclusions: This statement provides a roadmap for the investigation of SABV in preclinical models. This will increase the applicability of findings to both sexes, uncover sex-biased mechanisms in lung diseases, and identify novel therapeutic targets.

Background: Myocardial ischemia-reperfusion injury (IRI) arises by abrupt myocardial blood flow restoration after ischemia leading to cardiomyocyte dysfunction and death. Mechanisms of myocardial IRI involve an interplay of metabolic dysfunction, including branched-chain amino acid (BCAAs) imbalance, and mitochondrial dysfunction, resulting in oxidative stress, inflammation, and cardiomyocyte death. Our lab was the first to demonstrate the cardioprotective effects of intralipid (ILP), a safe lipid emulsion, against myocardial IRI in rodents; However, underlying mechanisms remain unclear. Research Hypothesis: We hypothesize that ILP protects IRI by restoring the depletion of BCAAs, thereby attenuating inflammation, oxidative stress, and apoptosis. Methods: Male rats were subjected to sham or IRI by LAD ligation for 30 min followed by 180 min of reperfusion. The IRI group received either a single bolus of ILP (20%, 5ml/kg body weight) or saline at the onset of reperfusion. RNA seq and LC Mass spectrometry were performed on left ventricle (LV). In vitro, H9C2 cardiac myoblasts were exposed to 3h of hypoxia followed by 6h of reoxygenation, with or without 20% ILP during reoxygenation. Seahorse assessed H9C2 mitochondrial function and RT-PCR quantified inflammatory and apoptotic markers expression. Results: LV transcriptomic analysis revealed that IRI significantly downregulated oxidative phosphorylation and fatty acid oxidation, while upregulating glycolysis, hypoxia, inflammation, and apoptosis pathways compared to sham. LV from ILP-treated rats exhibited similar pathway dynamics to levels observed in sham rats. In vitro, ILP administration during reoxygenation significantly enhanced ATP production and oxygen consumption in H9C2 cells compared to controls. Also, ILP-treated H9C2 cells exhibited upregulated expression of respective anti-apoptotic and antioxidant markers Bcl-2 and Sod2, downregulated expression of pro-apoptotic marker Bax, and pro-inflammatory TNA-α. Metabolomic profiling revealed that BCAAs including valine, leucine, and isoleucine were among the downregulated LV metabolites by IRI, but upregulated by ILP treatment upon IRI, with significant changes observed specifically for valine. Conclusion(s): Together, our data suggest that ILP administration at the onset of myocardial reperfusion preserves reduced BCAAs levels caused by IRI and improves mitochondrial function, leading to attenuated cardiomyocyte inflammation, apoptosis and oxidative stress.

Glioblastoma (GBM) is the deadliest brain cancer in adults, and all patients succumb to the tumor. While surgery followed by chemoradiotherapy delays disease progression, these treatments do not lead to tumor control, and targeted therapies or biologics have failed to further improve survival. Utilizing a transient radiation-induced state of multipotency, we used the adenylcyclase activator forskolin to alter the fate of irradiated glioma cells. The effects of the combined treatment on neuronal marker expression, cell cycle distribution, and proliferation were studied. Gene expression profiling was conducted using bulk RNA-seq. Changes in cell populations were investigated using single-cell RNA-seq. Effects on glioma stem cells (GSCs) were studied in extreme limiting dilution assays, and the effects on median survival were studied in both syngeneic and PDOX mouse models of GBM. The combined treatment induced the expression of neuronal markers in glioma cells, reduced proliferation, and led to a distinct gene expression profile. scRNA-seq revealed that the combined treatment forced glioma cells into a microglia- and neuron-like phenotype. In vivo, this treatment led to a loss of GSCs and prolonged median survival. Collectively, our data suggest that revisiting a differentiation therapy with forskolin in combination with radiation could lead to clinical benefit.

Pulmonary arterial hypertension (PAH) is marked by progressive vascular remodeling, but key cell type–specific regulatory programs driving this process remain poorly defined. We performed single-nucleus RNAseq of 67 PAH and donor lungs to generate the largest cell atlas of the human PAH lung to date, capturing all major vascular, immune, and stromal subpopulations. Myofibroblasts were significantly expanded in PAH and enriched for epithelial-to-mesenchymal transition and PAH-associated genetic variants. Ligand–receptor interaction and latent factor analysis identified a signaling program enriched for endothelial–stromal crosstalk and TGFβ signaling, correlating with histologic measures of remodeling. Trajectory analysis revealed that adventitial fibroblasts had the highest developmental potential among stromal cells. In a focused trajectory analysis modeling their transition to myofibroblasts, ITGA9 emerged as a top driver gene, with expression correlating with clinical severity. Spatial transcriptomics confirmed ITGA9 expression in myofibroblasts within remodeled vessels. ChIPseq demonstrated SMAD3 and SP1 binding at the ITGA9 promoter, supporting transcriptional regulation by TGFβ–related pathways. ITGA9 was elevated in PAH across independent datasets, including bulk lung tissue, animal models, and peripheral blood. Regulatory variants linked to ITGA9 expression in myofibroblasts also correlated with disease severity. These findings from an unbiased atlas-scale single-cell analysis of the PAH lung identify ITGA9 as a transcriptionally regulated driver of fibroblast remodeling, and nominate it as a potential biomarker and therapeutic target in PAH.

The effect of the gut microbiota extends beyond their habitant place from the gastrointestinal tract to distant organs, including the cardiovascular system. Research interest in the relationship between the heart and the gut microbiota has recently been emerging. The gut microbiota secretes metabolites, including Trimethylamine N-oxide (TMAO), short-chain fatty acids (SCFAs), bile acids (BAs), indole propionic acid (IPA), hydrogen sulfide (H2S), and phenylacetylglutamine (PAGln). In this review, we explore the accumulating evidence on the role of these secreted microbiota metabolites in the pathophysiology of ischemic and non-ischemic heart failure (HF) by summarizing current knowledge from clinical studies and experimental models. Elevated TMAO contributes to non-ischemic HF through TGF-ß/Smad signaling-mediated myocardial hypertrophy and fibrosis, impairments of mitochondrial energy production, DNA methylation pattern change, and intracellular calcium transport. Also, high-level TMAO can promote ischemic HF via inflammation, histone methylation-mediated vascular fibrosis, platelet hyperactivity, and thrombosis, as well as cholesterol accumulation and the activation of MAPK signaling. Reduced SCFAs upregulate Egr-1 protein, T-cell myocardial infiltration, and HDAC 5 and 6 activities, leading to non-ischemic HF, while reactive oxygen species production and the hyperactivation of caveolin-ACE axis result in ischemic HF. An altered BAs level worsens contractility, opens mitochondrial permeability transition pores inducing apoptosis, and enhances cholesterol accumulation, eventually exacerbating ischemic and non-ischemic HF. IPA, through the inhibition of nicotinamide N-methyl transferase expression and increased nicotinamide, NAD+/NADH, and SIRT3 levels, can ameliorate non-ischemic HF; meanwhile, H2S by suppressing Nox4 expression and mitochondrial ROS production by stimulating the PI3K/AKT pathway can also protect against non-ischemic HF. Furthermore, PAGln can affect sarcomere shortening ability and myocyte contraction. This emerging field of research opens new avenues for HF therapies by restoring gut microbiota through dietary interventions, prebiotics, probiotics, or fecal microbiota transplantation and as such normalizing circulating levels of TMAO, SCFA, BAs, IPA, H2S, and PAGln.

Background: Myocardial ischemia-reperfusion injury (IRI) refers to the tissue damage that occurs when blood supply returns to the heart after a period of ischemia and the sudden reintroduction of oxygen and nutrients paradoxically triggers a cascade of inflammation and cell death in both cardiomyocytes and coronary artery endothelial cells (EC). Our lab was the first to show Intralipid (ILP), the first safe lipid emulsion for human use, confers robust cardioprotection against IRI in rodents. However, the full downstream mechanisms remain unknown. Hypothesis: We hypothesize that ILP attenuates IRI-induced inflammation, and apoptosis in the myocardium via downregulation of N-myc downstream regulated gene 1 (NDRG1). Methods: We performed RNA Sequencing on the left ventricle (LV) of male Sprague-Dawley rats subjected to LAD ligation to induce IRI (30 min ischemia, 180 min reperfusion) or sham surgery. Rats received either one bolus of ILP (20% IV) or saline 5 minutes before the onset of reperfusion. Pathway enrichment analysis was performed using transcriptomics data. We performed qPCR to validate our transcriptomics data. We subjected H9C2 cardiac myoblasts and coronary artery endothelial cells (EC) to hypoxia (4h)/reoxygenation (2h) and ILP (20%) treatment during reoxygenation. We tested the functional role of NDRG1 in EC by silencing NDRG1 via siRNA. Results: Our transcriptomic pathway enrichment analysis revealed hypoxia and inflammatory response genes were significantly increased in the LV of rats subjected to IRI compared to sham but showed no significant difference between ILP-treated IRI hearts and sham controls. Our transcriptomics analysis identified seven significantly differentially-expressed genes, including the hypoxia molecular switch N-myc downstream-regulated gene 1 (NDRG1). We validated that NDRG1 mRNA in the LV in rats is upregulated by IRI and attenuated by ILP treatment at the onset of reperfusion to a similar level as sham rats. Our in-vitro data also showed that ILP significantly reduced hypoxia-reoxygenation induced upregulation of NDRG1 transcript levels in H9C2 and EC. Functionally, knockdown of NDRG1 in coronary EC using siNRGD1 reduced markers of EC activation (ICAM1), inflammation (IL-6) and apoptosis (cleaved caspase 3) compared to control siRNA. Conclusion: Taken together, our data suggests that ILP confers cardioprotection against IRI via downregulation of NDRG1 expression, leading to reduced inflammation, and apoptosis.

Background: Pulmonary hypertension (PH) is a fatal pulmonary vascular disease characterized by progressive increase in pulmonary arterial pressure, which leads to right ventricular failure and death. Pulmonary artery endothelial cell (PAEC) dysfunction and smooth muscle cell (PASMC) proliferation are two major hallmarks of PH, which induce inflammation and pulmonary vascular remodeling. The role of oxidized lipids in the pathogenesis of PH has been emerging, and our lab was the first to show that a diet enriched in a single oxidized fatty acid, 15-Hydroxyeicosatetraenoic Acid (15-HETE), is sufficient to cause PH in wild-type mice. However, the molecular mechanism underlying the causal role of dietary oxidized lipids in the development of PH remained unknown. Research Hypothesis: We hypothesize that dietary 15-HETE causes PH by promoting Stearoyl-CoA Desaturase-1 ( Scd1 ) mediated PAEC and PASMC dysfunction. Approach: C57BL/6 mice were fed with 15-HETE diet for three weeks to develop PH. We utilized RNA-Seq analysis on FACS-isolated enterocytes, from control and 15-HETE diet-fed mice to identify differences in genes involved in lipid metabolism. Lipidyzer™ analysis was used to measure the level of lipids in plasma. In vitro experiments were performed using human PAEC and PASMC. Results: Mice fed a 15-HETE-enriched diet for 3 weeks developed PH as right ventricular systolic pressure was significantly higher than chow-fed controls. RNA-seq of FACS-isolated enterocytes identified Scd1 as a novel gene that its expression is significantly upregulated in PH mice on 15-HETE diet compared to chow diet. The levels of Scd1 products, palmitoleate and oleate, were significantly increased in the plasma of 15-HETE mice. Mechanistically, Scd 1 product, Oleate was sufficient to increase expression of Scd1 and inflammatory marker IL-1β in PAEC, but not in PASMC. However, conditioned medium from Oleate-stimulated PAEC was able to increase IL-1β expression in PASMC and promote PASMC proliferation. Notably, EC-specific Scd1 Knock-Out mice did not develop PH on a 15-HETE diet, further highlighting the role of Scd 1 in promoting EC dysfunction in PH. Conclusion(s): Together, our data suggest that a 15-HETE-enriched diet induces upregulation of Scd1 in enterocytes, leading to the release of its products, oleate and palmitoleate, into the systemic circulation. These lipids promote PAEC dysfunction, which, in a paracrine manner, promote PASMC inflammation and proliferation.

Accumulating evidence suggests that dietary factors play a significant role in the pathogenesis of cardiovascular diseases, including PAH. Our group were the first to establish that a diet enriched in a single oxidized fatty acid, 15HETE, is sufficient to cause pulmonary hypertension (PH) in wild type mice. Our recent published work demonstrated that 15-HETE diet induces pulmonary hypertension by triggering pulmonary arterial endothelial cell (EC) apoptosis. However, the precise molecular mechanisms driving these processes remain unclear. We now demonstrate that 15HETE diet alters the intestinal flora of mice, decreasing beneficial bacteria that produce short-chain fatty acids (SCFAs) including propionate and butyrate. The reduction in SCFA producing bacteria was associated with decreased levels of propionate in the plasma of 15HETE diet fed mice as well as in our UCLA cohort PAH patients compared to control subjects. Supplementation of 15HETE diet with SCFA (propionate or butyrate) in the drinking water effectively prevented and even rescued 15-HETE induced PH. RNA-Seq analysis identified Stearoyl-CoA 9-desaturase (Scd1) as one of the differentially expressed genes in the lungs of 15-HETE diet fed mice compared to control mice. SCD1 is an enzyme that catalyzes the formation of monounsaturated fatty acids (MUFAs) including oleic acid. SCFA supplementation reduced Scd1 expression and serum oleate levels along with preventing 15-HETE induced PH. We show that treating pulmonary arterial EC (PAEC) with oleate is sufficient to promote inflammatory cytokine productions of IL-6 and IL-1b in PAEC. Furthermore, EC-specific Scd1 knock out develop significantly less PH in the 15-HETE diet model compared to control littermates. Taken together, our results demonstrate that dietary 15-HETE induces (i) dysbiosis in the small intestine luminal microbiome that results in the reduction of specific beneficial SCFA producing bacteria and ii) Scd1 gene expression and MUFA that promote pulmonary arterial endothelial cell dysfunction. Dietary supplementation of SCFA or EC-specific knockdown of Scd1 are sufficient to prevent 15HETE diet-induced PH. Our results offer novel promising approaches for PAH management.

Background: Pulmonary arterial hypertension (PAH) is a progressive and fatal disease characterized by pulmonary vascular remodeling, increased right ventricular pressure, and eventual heart failure. Elevated levels of oxidized lipid metabolites especially hydroxyeicosatetraenoic acids (HETEs) have been observed in both human patients and animal models, implicating these lipids in the disease’s pathogenesis. We previously demonstrated that dietary supplementation with 15-HETE for 3 weeks is sufficient to induce pulmonary hypertension (PH) in C57BL6/J. However, the molecular mechanisms remain poorly understood. During our investigations, we discovered that dietary 15-HETE increases systemic oxylipin levels of not only 15HETE but also other HETEs (including 5-, 12-), like PAH patients. We therefore hypothesized that 15-HETE promotes PH through an initial lipoxygenase (LOX)-mediated oxylipin amplification, which in turn drives metabolic and immune alterations in the gut and contribute to progression of PH. Methods: PH development was assessed by pulmonary arterial acceleration time (PAAT) and right ventricular systolic pressure (RVSP). LOX involvement was tested by blocking total LOX activity, treating 12/15-LOX-deficient mice with a 5-LOX inhibitor (zileuton). Plasma lipid profiles were analyzed mass spectrometry. Cytokine levels were measured by multiplex immunoassay. Flow cytometry was used to evaluate gut immune populations. Results: 15-HETE alone, 5-HETE alone or combined 5-HETE and 12-HETE supplementation induced PH, reflected by significantly decreased PAAT and elevated RVSP. Lipidomics analysis revealed increased oxilipins in 15-HETE treated mice. Elevation in plasma inflammatory cytokines (including eotaxin, G-CSF, IL-15, and MCP-1) and shifts in gut immune cells (increased Type 1 macrophages and decreased neutrophils) were observed in 15-HETE treated mice, suggesting an inflammatory response initiated in the gut. Finally, 12/15-LOX deficiency combined with zileuton treatment not only reduced intestinal 5-, 12-, 15-HETE levels but also prevented 15-HETE induced PH suggesting that 15-HETE mediated oxylipin amplification is causal in the development of PH in mice treated with 15-HETE. Conclusions: Our findings suggest that the gut appears to play a previously underappreciated role in the development of PH, highlighting the potential of targeting LOX pathways and gut–lung interactions in the treatment of PAH.

Pulmonary arterial hypertension (PAH) is a severe, progressive disease characterized by elevated pulmonary artery pressure, ultimately leading to right heart failure. Key contributors to PAH include endothelial cell (EC) dysfunction and inflammation, influenced in part by oxidized lipids and diminished expression of the low-density lipoprotein receptor (LDLR). PAH patients exhibit reduced LDLR in lung tissue, alongside elevated levels of oxidized lipids and inflammatory markers. Studies using Western diet (WD)-fed Ldlr-/- mice indicate that pulmonary hypertension (PH) can precede left ventricular dysfunction, highlighting the role of lipid oxidation. Additionally, WD consumption leads to gut microbiome dysbiosis, which reduces beneficial short-chain fatty acid (SCFA)-producing bacteria, decreases butyrate levels, and promotes systemic inflammation, potentially worsening PAH. Thus, lipid dysregulation, LDLR downregulation, and SCFA reduction offer new insights into the pathogenesis of PAH; however, the specific mechanisms through which these factors interact and contribute to the PAH progression remain largely unknown. RNA-Seq analysis of FACS sorted enterocytes from jejunum of Ldlr-/- mice fed a chow or a WD identified Stearoyl-CoA 9-desaturase (Scd1) as the most significantly WD upregulated gene. Scd1 encodes an enzyme involved in fatty acid biosynthesis, primarily the synthesis of oleic acid. To investigate the role and mechanisms Scd1 in WD diet mediated PH, we developed enterocyte specific Scd1 knockout mice (Scd1fl/fl/VilCre/Ldlr-/-). Scd1fl/fl/Ldlr-/- and Scd1fl/fl/VilCre/Ldlr-/-mice aged 8 to 12 weeks (n=8 per group) were fed either a chow diet or WD for two weeks. At the end of the study, serum levels of markers of systemic inflammation (endotoxin, SAA, IL-6) and jejunal levels of fatty acids including oleate and SCFA were determined. Enterocyte knockdown of Scd1 had lower levels of oleate in both the jejunum and serum and prevented the elevation of WD mediated markers of systemic inflammation. Moreover, enterocyte knockdown of Scd1 prevented WD mediated loss of butyrate levels in the jejunum tissue. Our results underscore the critical role of Scd1 in the development of pulmonary hypertension and suggest that the WD mediated upregulation of Scd1 in enterocytes contributes to gut dysbiosis and reduced SCFA production, particularly butyrate, exacerbating systemic inflammation and PH.

Introduction: Pulmonary arterial hypertension (PAH) is a chronic lung disease characterized by progressive increase in pulmonary arterial pressure leading to right ventricular (RV) failure and death. The molecular mechanisms causal in the development of PAH are not well understood. Recently, we demonstrated that oral administration of an oxidized lipid, 15-HETE (elevated in human PAH), is sufficient to cause PH in wild type C57BL/6 mice. Using this new model of PH, we conducted multi-omics analyses to identify novel pathways and mechanisms that participate in the development of PH. Methods/Results: 16S rRNA sequencing of the feces revealed that dietary 15-HETE causes dysbiosis, including a significant reduction in the abundance of short chain fatty acid (SCFA) producing bacteria. Supplementing drinking water of 15-HETE fed mice with 0.1M sodium butyrate (SB) attenuated the development of PH. Plasma metabolomics identified a decrease in cholesterol sulfate (CS) of PH mice that was reversed by SB. Using STITCH, we identified two genes connected to CS namely, RAR-related orphan receptor A (RORA) and RORC. RORA is a direct effector of nuclear receptor corepressor 1 (NCOR1), which we identified in RNA-seq data sets from both the jejunum and enterocytes of PH mice. We confirmed our findings by qPCR and demonstrated that NCOR1 expression in the jejunum of mice fed 15-HETE is decreased while SB supplementation prevented the decrease, mirroring the levels of CS. Conclusion: Using multiomics data from microbiome, metabolome, and transcriptome, we identified a novel pathway; butyrate-RORA-NCOR1-cholesterol sulfate, that participates in the development of PH in mice. This research is supported by T32HL144449, UCLA and Caltech integrated Cardiovascular Medicine for Bioengineers (iCMB) and by the National Heart, Lung, and Blood Institute grant 1R01HL162124-02.

Maternal mortality rates are at an all-time high across the world and are set to increase in subsequent years. Cardiovascular disease is the leading cause of death during pregnancy and postpartum, especially in the United States. Therefore, understanding the physiological changes in the cardiovascular system during normal pregnancy is necessary to understand disease-related pathology. Significant systemic and cardiovascular physiological changes occur during pregnancy that are essential for supporting the maternal-fetal dyad. The physiological impact of pregnancy on the cardiovascular system has been examined in both experimental animal models and in humans. However, there is a continued need in this field of study to provide increased rigor and reproducibility. Therefore, these guidelines aim to provide information regarding best practices and recommendations to accurately and rigorously measure cardiovascular physiology during normal and cardiovascular disease-complicated pregnancies in human and animal models.

BACKGROUND: Integrative multiomics can elucidate pulmonary arterial hypertension (PAH) pathobiology, but procuring human PAH lung samples is rare. METHODS: We leveraged transcriptomic profiling and deep phenotyping of the largest multicenter PAH lung biobank to date (96 disease and 52 control) by integration with clinicopathologic data, genome-wide association studies, Bayesian regulatory networks, single-cell transcriptomics, and pharmacotranscriptomics. RESULTS: We identified 2 potentially protective gene network modules associated with vascular cells, and we validated ASPN , coding for asporin, as a key hub gene that is upregulated as a compensatory response to counteract PAH. We found that asporin is upregulated in lungs and plasma of multiple independent PAH cohorts and correlates with reduced PAH severity. We show that asporin inhibits proliferation and transforming growth factor–β/phosphorylated SMAD2/3 signaling in pulmonary artery smooth muscle cells from PAH lungs. We demonstrate in Sugen-hypoxia rats that ASPN knockdown exacerbated PAH and recombinant asporin attenuated PAH. CONCLUSIONS: Our integrative systems biology approach to dissect the PAH lung transcriptome uncovered asporin as a novel protective target with therapeutic potential in PAH.

Pulmonary arterial hypertension (PAH) is a severe disease caused by progressive distal pulmonary artery obstruction. One cause of PAH are loss-of-function mutations in the potassium channel subfamily K member 3 (KCNK3). KCNK3 encodes a two-pore domain potassium channel, which is crucial for pulmonary circulation homeostasis. However, our understanding of the pathophysiological mechanisms underlying KCNK3 dysfunction in PAH is still incomplete. Taking advantage of unique Kcnk3-deficient rats, we analyzed the transcriptomic changes in the lungs from homozygous Kcnk3-deficient rats and wild-type (WT) littermates and compared them to PAH patient transcriptomic data. Transcriptome analysis of lung tissue obtained from WT and Kcnk3-deficient rats identified 1915 down- or upregulated genes. In addition, despite limited similarities at the gene level, we found a strong common signature at the pathway level in PAH patients and Kcnk3-deficient rat lungs, especially for immune response. Using the dysregulated genes involved in the immune response, we identified Spleen Associated Tyrosine Kinase (SYK), a significantly downregulated gene in human PAH patients and Kcnk3-deficient rats, as a hub gene. Our data suggests that the altered immune system response observed in PAH patients may be partly explained by KCNK3 dysfunction through the alteration of SYK expression.

Integrative multiomics can help elucidate the pathophysiology of pulmonary fibrosis (PF)-associated pulmonary hypertension (PH) (PF-PH). Weighted gene coexpression network analysis (WGCNA) was performed on a transcriptomic dataset of explanted lung tissue from 116 patients with PF. Patients were stratified by pulmonary vascular resistance (PVR), and differential gene expression analysis was conducted. Gene modules were correlated with hemodynamics at the time of transplantation and tested for enrichment in the lung transcriptomics signature of an independent pulmonary arterial hypertension (PAH) cohort. We found 1,250 differentially expressed genes between high and low PVR groups. WGCNA identified that black and yellowgreen modules negatively correlated with PVR, whereas the tan and darkgrey modules are positively correlated with PVR in PF-PH. In addition, the tan module showed the strongest enrichment for an independent PAH gene signature, suggesting shared gene expression patterns between PAH and PF-PH. Pharmacotranscriptomic analysis using the Connectivity Map implicated the tan and darkgrey modules as potentially pathogenic in PF-PH, given their combined module signature demonstrated a high negative connectivity score for treprostinil, a medication used in the treatment of PF-PH, and a high positive connectivity score for bone morphogenetic protein (BMP) loss of function. Pathway enrichment analysis revealed that inflammatory pathways and oxidative phosphorylation were downregulated, whereas epithelial-mesenchymal transition was upregulated in modules associated with increased PVR. Our integrative systems biology approach to the lung transcriptome of PF with and without PH identified several PH-associated coexpression modules and gene targets with shared molecular features with PAH warranting further investigation to uncover potential new therapies for PF-PH. NEW & NOTEWORTHY An integrative systems biology approach that included transcriptomic analysis of explanted lung tissue from patients with pulmonary fibrosis (PF) with and without pulmonary hypertension (PH) undergoing lung transplantation, combined with hemodynamic correlation and pharmacotranscriptomics, identified modules of genes associated with pulmonary vascular disease severity. Comparison with an independent pulmonary arterial hypertension (PAH) dataset identified shared gene expression patterns between PAH and PF-PH.