Cancer therapies are being considered for treating rare noncancerous diseases like pulmonary hypertension (PH), but effective computational screening is lacking. Via transcriptomic differential ...dependency analyses leveraging parallels between cancer and PH, we mapped a landscape of cancer drug functions dependent upon rewiring of PH gene clusters. Bromodomain and extra-terminal motif (BET) protein inhibitors were predicted to rely upon several gene clusters inclusive of galectin-8 (LGALS8). Correspondingly, LGALS8 was found to mediate the BET inhibitor–dependent control of endothelial apoptosis, an essential role for PH in vivo. Separately, a piperlongumine analog’s actions were predicted to depend upon the iron-sulfur biogenesis gene ISCU. Correspondingly, the analog was found to inhibit ISCU glutathionylation, rescuing oxidative metabolism, decreasing endothelial apoptosis, and improving PH. Thus, we identified crucial drug-gene axes central to endothelial dysfunction and therapeutic priorities for PH. These results establish a wide-ranging, network dependency platform to redefine cancer drugs for use in noncancerous conditions.
Utilizing publicly available ribonucleic acid sequencing data, we identified
as a BMPR2-related gene differentially expressed between induced pluripotent stem cell-endothelial cells derived from ...pulmonary arterial hypertension (PAH) patients carrying pathogenic BMPR2 mutations and control patients without mutations. Endothelial SCUBE1 expression was decreased by known triggers of PAH, and its down-regulation recapitulated known BMPR2-associated endothelial pathophenotypes in vitro. Meanwhile, SCUBE1 concentrations were reduced in plasma obtained from PAH rodent models and patients with PAH, whereas plasma concentrations were tightly correlated with hemodynamic markers of disease severity. Taken together, these data implicate SCUBE1 as a novel contributor to PAH pathogenesis with potential therapeutic, diagnostic, and prognostic applications.
Full text
Available for:
GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Cancer therapies are being considered for treating rare noncancerous diseases like pulmonary hypertension (PH), but effective computational screening is lacking. Via transcriptomic differential ...dependency analyses leveraging parallels between cancer and PH, we mapped a landscape of cancer drug functions dependent upon rewiring of PH gene clusters. Bromodomain and extra-terminal motif (BET) protein inhibitors were predicted to rely upon several gene clusters inclusive of galectin-8 (LGALS8). Correspondingly, LGALS8 was found to mediate the BET inhibitor-dependent control of endothelial apoptosis, an essential role for PH in vivo. Separately, a piperlongumine analog's actions were predicted to depend upon the iron-sulfur biogenesis gene ISCU. Correspondingly, the analog was found to inhibit ISCU glutathionylation, rescuing oxidative metabolism, decreasing endothelial apoptosis, and improving PH. Thus, we identified crucial drug-gene axes central to endothelial dysfunction and therapeutic priorities for PH. These results establish a wide-ranging, network dependency platform to redefine cancer drugs for use in noncancerous conditions.
Display omitted
•Guided by public sequencing data of iPSC-derived BMPR2-mutant ECs derived from patients with PAH and related family members, SCUBE1 was found to be differentially expressed in ECs ...carrying BMPR2 mutations, dependent on HIF1A and down-regulated by PAH triggers.•SCUBE1 deficiency controlled BMPR2-associated SMAD1/5/9 signaling, thus regulating endothelial angiogenic potential, proliferation, and apoptosis.•SCUBE1 was decreased specifically in plasma and lungs in rodents and patients with PAH but not in those with PH due to left heart disease or other acute or chronic pulmonary and cardiovascular diseases.•A plasma SCUBE1 cutpoint of 5.46 ng/ml was defined to distinguish PAH from non-PAH contexts, with a high specificity (0.87) and a significant diagnostic OR (7.6).•In PAH patients, plasma SCUBE1 levels negatively correlated with PA pressure, pulmonary vascular resistance, and RV dysfunction.•These findings support the notion of SCUBE1 as a clinical biomarker of PAH, reflecting the severity and progression of PAH and inherently linked to disease pathogenesis and genetic predisposition.
Utilizing publicly available ribonucleic acid sequencing data, we identified SCUBE1 as a BMPR2-related gene differentially expressed between induced pluripotent stem cell-endothelial cells derived from pulmonary arterial hypertension (PAH) patients carrying pathogenic BMPR2 mutations and control patients without mutations. Endothelial SCUBE1 expression was decreased by known triggers of PAH, and its down-regulation recapitulated known BMPR2-associated endothelial pathophenotypes in vitro. Meanwhile, SCUBE1 concentrations were reduced in plasma obtained from PAH rodent models and patients with PAH, whereas plasma concentrations were tightly correlated with hemodynamic markers of disease severity. Taken together, these data implicate SCUBE1 as a novel contributor to PAH pathogenesis with potential therapeutic, diagnostic, and prognostic applications.
Full text
Available for:
GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
BackgroundPulmonary arterial hypertension (PAH) is an enigmatic vascular disorder characterized by pulmonary vascular remodeling and eventual right heart failure. A genome-wide association study ...(GWAS) on a cohort of PAH patients identified SNPs in the nuclear receptor co-activator 7 (NCOA7) gene that were associated with increased patient survival. Limited studies have suggested that NCOA7 has an important role in innate immunity. When coupled with the increasing appreciation of immune dysregulation as an emerging driver of vascular remodeling in PAH, NCOA7 is implicated as an appealing candidate to investigate.HypothesisInduction of NCOA7 expression, via either proinflammatory or genetic triggers, results in endothelial cell (EC) dysfunction and PAH.Methods and ResultsHuman pulmonary arterial endothelial cells (HPAECs) demonstrated marked upregulation of NCOA7 (33.01 ± 2.80, P<0.001) when challenged with IL-1β—a cytokine elevated in the serum of PAH patients. In addition, a multitude of PAH-associated genetic triggers (eg, ACRVL1, BMPR2, EIF2AK4, & ENG) upregulated NCOA7 expression. RNAi of NCOA7 abrogated IL-1β-mediated apoptosis (-4.24 ± 0.25, P<0.0001) and enhanced the angiogenic capacity of HPAECs via in vitro tube formation. Transcriptomic analysis of NCOA7-deficient HPAECs revealed significant enrichment for cholesterol metabolism-related processes, namely the marked shutdown of de novo cholesterol synthesis through the enzyme lanosterol-14-α-demethylase (CYP51A1) that initiates the pathway of converting lanosterol into cholesterol. Both the RNAi of CYP51A1 and exogenous application of lanosterol similarly attenuated IL-1β-mediated apoptosis (-2.33 ± 0.18, P<0.0001 and -1.55 ± 0.26, P<0.01, respectively) and enhanced angiogenic potential.ConclusionsVarious known PAH triggers induce the expression of NCOA7, suggestive of a potential centrality at the nexus of an immune and genetic interface. Moreover, deficiency of NCOA7 prevents EC apoptosis and enhances angiogenic capacity, perhaps through the inhibition of CYP51A1 and subsequent accumulation of lanosterol. These data demonstrate a novel role for sterol homeostasis in mediating EC behavior and suggest that NCOA7 deficiency may be protective in PAH.
BackgroundDysregulation of specific microRNAs promotes pulmonary hypertension (PH). MiR-210 is one such microRNA that is induced by hypoxia and regulates mitochondrial targets to repress oxidative ...respiration in pulmonary endothelial cells. Recently, it has been proposed that the vascular pathobiology of microRNAs may rely on their transport in the blood to the vascular bed, through either vascular infiltration of interstitial lung macrophages or extracellular microvesicles in plasma. However, whether the development of PH depends critically on blood-borne transport of pathogenic microRNAs remains unanswered.Methods & ResultsTo interrogate the role of blood-borne miR-210 in PH pathogenesis, we utilized in vivo parabiosis (where circulatory systems of two mice were surgically connected over 4 weeks) in the context of miR-210+/+ (WT) and miR-210-/- (KO) mice. One-week post-surgery, mouse pairs were exposed to 10% O2 for 3 weeks to induce miR-210 up-regulation. KO-KO pairs were protected from PH compared with WT-WT pairs, as displayed by lower RVSP (26.4±0.5 mmHg, n=7 vs. 30.3±1.2 mmHg, n=10, p<0.05, mean±SEM). Importantly, in WT-KO pairs, the KO mouse developed higher RVSP versus KO-KO pairs (30.8±1.2 mmHg, n=9 vs. 26.4±0.5 mmHg, n=7, p<0.01). Furthermore, by flow sorting (FACS), an expansion of CD64/CD11b-positive interstitial lung macrophages was observed in KO mice of WT-KO pairs versus KO-KO partners (5176.7/mg tissue vs. 2132.5/mg tissue, n=3, p<0.05). To determine if endogenous blood-borne miR-210 can be delivered to the vasculature in vivo, miR-210 expression was quantified by RT-qPCR in plasma and in isolated CD31-positive lung endothelial cells. While miR-210 was not detectable in paired KO mice, in WT-KO pairs, extracellular plasma miR-210 was robustly expressed in KO and WT partners (0.71±0.1 fold change vs. 1.03±0.4 fold change, n=3, p>0.05), comparable with WT-WT pairs (1.01±0.2 fold change, n=3). Moreover, in the KO partner of WT-KO pairs, intracellular miR-210 in CD31-positive lung endothelial cells was observed at substantial levels, albeit lower than in lung endothelial cells of the WT partners (1.04±0.1 fold change vs. 69.0±8.1 fold change, n=10, p<0.0001).ConclusionsBlood-borne transport of miR-210 with consequent uptake into pulmonary vascular endothelial cells promotes PH in vivo. These findings offer evidence of a pathogenic endocrine function of microRNAs in PH, thus revealing a surprising level of multi-system, RNA-mediated crosstalk in anatomic compartments beyond the cardiopulmonary space.
BackgroundPulmonary hypertension (PH) is a deadly vascular disease with poorly defined molecular origins. While evidence is emerging in regard to dysregulation of long non-coding RNAs (lncRNAs) in ...PH, the exact pathobiology of these molecules in the pulmonary vasculature is unknown.HypothesisSpecific lncRNAs control the dysregulated metabolic and proliferative states of endothelial cells, leading to the development of PH.Methods and ResultslncRNA 5031425E22RIK (E22), which mapped to a human ortholog KMT2E-Antisense RNA1 (KMT2E-AS1), was identified by RNA sequencing to be differentially expressed and upregulated in PH mouse lung tissue (1.79 fold change ± 0.49, p<0.05). Across mammals, this lncRNA sits head-to-head adjacent to the chromosomal location of histone lysine N-methyltransferase 2E gene (KMT2E), a family member of regulators controlling histone 3 lysine 4 trimethylation (H3K4Me3) and chromatin remodeling. In multiple in vivo rodent and human models of PH, both mouse E22 (1.41 fold change ± 0.09, p<0.05) and human KMT2E-AS1 (2.13 fold change ± 0.32, p<0.05) were increased. Via computational analyses and biochemical experimental approaches studying RNA/protein interactions and gain/loss-of-function methods of modulating this lncRNA in pulmonary arterial endothelial cells in vitro, KMT2E-AS1 was found to be a hypoxia-driven RNA that complexes with KMT2E protein to alter H3K4Me3. These epigenetic events up-regulate HIF-2α expression and activity, and inhibit downstream oxidative phosphorylation, thus increasing endothelial proliferation and vasoconstriction and decreasing apoptosis. In vivo manipulation of mouse E22 via genetic deletion of the 500 bp conserved sequence of E22 as well as virus-mediated overexpression of E22 in mouse pulmonary vasculature demonstrated that E22 is necessary and sufficient to promote PH via vascular muscularization and worsen hemodynamics.ConclusionKMT2E-AS1 serves as a master epigenetic regulator of endothelial metabolic reprogramming in hypoxia with conserved activity in rodents and humans for promoting PH. These results provide implications in identifying a long non-coding RNA as a crucial epigenetic and metabolic regulator of PH, potentially acting as a novel therapeutic target.
BackgroundFrataxin (FXN) controls iron-sulfur (Fe-S) biogenesis and DNA integrity. Mutations cause Friedreich’s ataxia (FRDA) and hypertrophic cardiomyopathy, often complicated by pulmonary ...hypertension (PH), a deadly disease with a puzzling progression of endothelial phenotypes. While DNA damage leads to endothelial senescence in other vascular diseases, whether endothelial FXN deficiency and its genotoxic effects predispose to multiple subtypes of PH remains unclear.HypothesisGenetic and acquired FXN deficiency induces endothelial evolution from DNA damage to senescence in PH.MethodsHuman pulmonary artery endothelial cells (PAECs) and inducible pluripotent stem cell-derived endothelial cells (iPSC-ECs) from FRDA patients were used. Cell-specific Fxn-/- mice and mice subjected to endothelial delivery of FXN RNAi were probed along with in vivo models of Group 1, 2, and 3 PH.ResultsRodent, nonhuman primate, and human models of Groups 1-3 PH displayed reduced FXN and elevated senescence marker p16. In PAECs, hypoxia (0.48-fold change ± 0.05, P<0.01, mean ± SD) and IL-1β (0.47-fold change ± 0.01, P<0.05) decreased FXN via HIF-α-dependent epigenetic modulation. RNA sequencing in FXN-deficient PAECs revealed cell cycle and DNA damage pathway enrichment (FDR<0.05). Acute FXN knockdown abrogated Fe-S biogenesis (0.57-fold change ± 0.05, P<0.01), increased BrdU integration and DNA damage-sensitive p21 but not cell number, signaling arrest. Sustained FXN depletion in PAECs and iPSC-ECs led to persistent p-γH2AX, a double-strand break marker reflecting DNA damage response, and eventual apoptosis resistance and enhanced p16, signifying senescence. FXN deficiency also impaired mitochondrial respiration, vasomotor relaxation, and angiogenesis. Notably, genetic (RVSP 32.43 mmHg ± 1.45 v 28.74 ± 0.44, P<0.05) or RNAi-based endothelial Fxn deficiency in hypoxic mice increased p-γH2AX and p16 and worsened PH but smooth muscle Fxn-/- mice did not.ConclusionsFXN deficiency drives pathogenic evolution of the endothelium in PH, culminating in senescence. Our data identify a unifying endothelial mechanism across PH subtypes, including Group 2 PH and FRDA patients, while emphasizing Fe-S-specific diagnostic and therapeutic targets.
BackgroundEndothelial iron-sulfur (Fe-S) cluster deficiency promotes pulmonary hypertension (PH), but the pathogenic mechanisms are incompletely defined. Frataxin (FXN) controls Fe-S cluster assembly ...and DNA damage; mutations cause Friedreich’s ataxia (FRDA) and hypertrophic cardiomyopathy, often accompanied by PH.HypothesisFXN deficiency, due to genetic or acquired triggers, disrupts endothelial Fe-S-dependent DNA replication and damage to promote PH.MethodsHuman pulmonary arterial endothelial cells (PAECs) and induced pluripotent stem cell-derived endothelial cells (iPSC-ECs) from FRDA patients were studied. RNA sequencing of long and short RNAs was performed along with gene set enrichment analysis (GSEA). Endothelial-specific FXN knockout mice and mice with FXN RNAi delivered to the endothelium were studied.ResultsIn PAECs, hypoxia (0.48-fold change ± 0.05, P<0.01, mean ± SEM) and IL-1β (0.47-fold change ± 0.01, P<0.05) decreased FXN. Inhibition of the epigenetic modulators BRD2/4 rescued expression. FXN knockdown by RNAi decreased Fe-S clusters (0.57-fold change ± 0.05, P<0.01), increased BrDU incorporation (2.49-fold change ± 0.07, P<0.01) and propidium iodide staining in S-phase (but not cell number), signifying S-phase arrest. Double-stranded DNA breaks increased, reflected by induction of the DNA damage response factor p-γH2AX. RNA sequencing and GSEA in FXN-deficient PAECs revealed 203 pathways related to DNA replication and damage, all containing differentially expressed genes inversely regulated by 27 reciprocally expressed microRNAs (FDR<0.05). Moreover, FRDA iPSC-ECs revealed elevated senescence markers, p16 and p21. FXN deficiency also increased vasomotor tone and decreased angiogenesis. Pharmacologic (RVSP 34.31 mmHg ± 0.18 v 29.77 ± 0.93, P<0.05) and genetic endothelial FXN deficiency in hypoxic mice promoted PH.ConclusionsGenetic or epigenetic endothelial FXN deficiency impairs Fe-S-mediated DNA synthesis and repair leading to DNA damage and potentially senescence, thus promoting PH. Our results emphasize FXN-specific endothelial replication stress as a novel pathobiologic phenotype in this disease, thus endorsing therapies that relieve genotoxic processes in PH.
Background and HypothesisDeficiencies of iron-sulfur (Fe-S) clusters, metal complexes that control redox state and mitochondrial metabolism, have been linked to pulmonary hypertension (PH), a deadly ...vascular disease with poorly defined molecular origins. The BolA Family Member 3 (BOLA3) regulates Fe-S biogenesis, and mutations in BOLA3 result in multiple mitochondrial dysfunction syndrome, a fatal disorder associated with PH. However, the exact mechanistic role of BOLA3 in PH remains undefined.Methods and ResultsIn cultured hypoxic PAECs as well as lung from human and multiple rodent models of PH, endothelial BOLA3 expression was down-regulated, which involved HIF-2α-dependent transcriptional repression via HDAC-mediated histone deacetylation. In vitro gain and loss of function studies demonstrated that BOLA3 regulated Fe-S integrity, thus modulating lipoate-containing 2-oxoacid dehydrogenases with consequent control over glycolysis and mitochondrial respiration. In addition, BOLA3 deficiency down-regulated the glycine cleavage system protein H (GCSH), thus bolstering intracellular glycine content. As a result of these alterations of oxidative metabolism and glycine levels, BOLA3 deficiency increased endothelial proliferation, survival, and vasoconstriction, while decreasing angiogenic potential. In vivo, pharmacologic knockdown of endothelial BOLA3 and targeted overexpression of BOLA3 demonstrated that BOLA3 deficiency promotes histologic and hemodynamic manifestations of PH.ConclusionIn aggregate, BOLA3 acts as a crucial lynchpin connecting Fe-S-dependent oxidative respiration and glycine homeostasis with endothelial metabolic re-programming critical to PH pathogenesis. These results provide a molecular explanation for the clinical associations linking PH with hyperglycemic syndromes and mitochondrial disorders. These findings also identify novel metabolic targets, including those involved in epigenetics, iron-sulfur biogenesis, and glycine biology, for diagnostic and therapeutic development in this disease.
PDF
