Introduction: Pulmonary hypertension (PH) is a fatal disease without a cure, in which endothelial dysfunction drives pathologic remodeling of the pulmonary vasculature. Individuals with Smith-Lemli-Opitz syndrome (SLOS) develop PH, but the underlying mechanisms remain undefined. SLOS is an autosomal recessive disorder of cholesterol synthesis, resulting from loss-of-function mutations in 7-dehydrocholesterol reductase (DHCR7)-the terminal enzyme in the de novo synthesis of cholesterol- that leads to accumulation of 7-dehydrocholesterol (7-DHC) and cytotoxic oxysterol species. The role of DHCR7 in PH, however, is unknown. Hypothesis: Elevated cytotoxic oxysterols resulting from genetic or acquired DHCR7 deficiency promotes endothelial dysfunction and the development of PH. Methods & Results: DHCR7 mRNA and protein expression was decreased in pulmonary artery endothelial cells (ECs) in cellular (P=0.0089), rodent, and human PH models, mirroring DHCR7 reduction in SLOS. Both genetic loss of DHCR7 and hypoxia promoted a global shutdown of de novo cholesterol synthesis, resulting in an accumulation of cholesterol intermediates and increased derivative oxysterols. Specifically, 7β-hydroxycholesterol (7β-HC) was elevated in DHCR7-deficient ECs (P<0.0001), and 7β-HC drove endothelial apoptosis (P<0.0001) via downregulation of insulin-like growth factor 1 (IGF1) in ECs (fold change=0.101 ± 0.034, FDR=6.65E-11). Conversely, overexpression of DHCR7 upregulated IGF1 expression (P<0.0007), and IGF1 delivery reversed apoptosis (P<0.0001). Correspondingly, ECs derived from SLOS patient stem cells with DHCR7 mutations (p.T93M/c.964-1G>C) exhibited IGF1-dependent apoptosis. Finally, EC-specific DHCR7 deletion in chronically hypoxic mice induced more severe PH in vivo (right ventricular systolic pressure 36.4 mmHg vs. 30.8 mmHg, P<0.0130). Conclusions: Resulting either from hypoxic or genetic causes, DHCR7 deficiency promoted EC apoptosis and PH via 7β-HC accumulation and subsequent IGF1 downregulation. Our work is the first to define an oxysterol-dependent mechanism underlying the link between genetic and acquired DHCR7 deficiency and PH.
Vascular inflammation critically regulates endothelial cell (EC) pathophenotypes, particularly in pulmonary arterial hypertension (PAH). Dysregulation of lysosomal activity and cholesterol metabolism have known inflammatory roles in disease, but their relevance to PAH is unclear. In human pulmonary arterial ECs and in PAH, we found that inflammatory cytokine induction of the nuclear receptor coactivator 7 (NCOA7) both preserved lysosomal acidification and served as a homeostatic brake to constrain EC immunoactivation. Conversely, NCOA7 deficiency promoted lysosomal dysfunction and proinflammatory oxysterol/bile acid generation that, in turn, contributed to EC pathophenotypes. In vivo, mice deficient for Ncoa7 or exposed to the inflammatory bile acid 7α-hydroxy-3-oxo-4-cholestenoic acid (7HOCA) displayed worsened PAH. Emphasizing this mechanism in human PAH, an unbiased, metabolome-wide association study (N=2,756) identified a plasma signature of the same NCOA7-dependent oxysterols/bile acids associated with PAH mortality (P<1.1x10-6). Supporting a genetic predisposition to NCOA7 deficiency, in genome-edited, stem cell-derived ECs, the common variant intronic SNP rs11154337 in NCOA7 regulated NCOA7 expression, lysosomal activity, oxysterol/bile acid production, and EC immunoactivation. Correspondingly, SNP rs11154337 was associated with PAH severity via six-minute walk distance and mortality in discovery (N=93, P=0.0250; HR=0.44, 95% CI [0.21-0.90]) and validation (N=630, P=2x10-4; HR=0.49, 95% CI [0.34-0.71]) cohorts. Finally, utilizing computational modeling of small molecule binding to NCOA7, we predicted and synthesized a novel activator of NCOA7 that prevented EC immunoactivation and reversed indices of rodent PAH. In summary, we have established a genetic and metabolic paradigm and a novel therapeutic agent that links lysosomal biology as well as oxysterol and bile acid processes to EC inflammation and PAH pathobiology. This paradigm carries broad implications for diagnostic and therapeutic development in PAH and in other conditions dependent upon acquired and innate immune regulation of vascular disease.
Isolated congenital aneurysms of the iliac artery are unusual. This report describes such a case, revealed by acute rupture and successfully operated on.
Background Pulmonary arterial hypertension (PAH) is a complex, fatal disease where disease severity has been associated with the single nucleotide polymorphism (SNP) rs2856830, located near the human leukocyte antigen DPA1 (HLA-DPA1) gene. We aimed to define the genetic architecture of functional variants associated with PAH disease severity by identifying allele-specific binding transcription factors and downstream targets that control endothelial pathophenotypes and PAH. Methods and Results Electrophoretic mobility shift assays of oligonucleotides containing SNP rs2856830 and 8 SNPs in linkage disequilibrium revealed functional SNPs via allele-imbalanced binding to human pulmonary arterial endothelial cell nuclear proteins. DNA pulldown proteomics identified SNP-binding proteins. SNP genotyping and clinical correlation analysis were performed in 84 patients with PAH at University of Pittsburgh Medical Center and in 679 patients with PAH in the
In this work an in-vitro flow experiment is conducted to elucidate the flow behavior in simplified aortic dissection (AD) disease geometries. In AD, the innermost layer of the aortic wall is locally and partially torn allowing blood to flow between the wall layers forming a parallel blood stream in what is known as the false lumen. The aim of this work is to elucidate the disease flow physics, and to provide guidance in diagnostic radiology, particularly contrast injected computed tomography (CT), where understanding flow patterns and mixing behavior is important for accurate diagnosis. In contrast-CT, dye is injected in the peripheral blood stream to illuminate the blood vessels and identify vascular abnormalities. The flow patterns and the dye transport dynamics impact the nature of the CT images and their interpretation. Particle image velocimetry (PIV) is used to quantify the AD flow fields, and laser-induced fluorescence (LIF) is implemented to visualize and assess the mixing behavior of dye in the false and true lumens. Interesting flow patterns are revealed and discussed in the context of their possible contribution to tear expansion and flapping, and to the elevated mean pressure in the false lumen that is reported in the literature.
Vascular inflammation regulates endothelial pathophenotypes, particularly in pulmonary arterial hypertension (PAH). Dysregulated lysosomal activity and cholesterol metabolism activate pathogenic inflammation, but their relevance to PAH is unclear. Nuclear receptor coactivator 7 ( NCOA7 ) deficiency in endothelium produced an oxysterol and bile acid signature through lysosomal dysregulation, promoting endothelial pathophenotypes. This oxysterol signature overlapped with a plasma metabolite signature associated with human PAH mortality. Mice deficient for endothelial Ncoa7 or exposed to an inflammatory bile acid developed worsened PAH. Genetic predisposition to NCOA7 deficiency was driven by single-nucleotide polymorphism rs11154337, which alters endothelial immunoactivation and is associated with human PAH mortality. An NCOA7-activating agent reversed endothelial immunoactivation and rodent PAH. Thus, we established a genetic and metabolic paradigm that links lysosomal biology and oxysterol processes to endothelial inflammation and PAH.
Background: Chronic thromboembolic pulmonary hypertension (CTEPH) is a rare and mysterious complication of pulmonary embolism, characterized by chronic pulmonary arterial thrombosis, occlusions and vasculopathy. Emerging evidence has suggested genetic relevance of CTEPH, particularly to ABO blood type. Genome-wide association study (GWAS) has identified ABO gene as the most significant genetic locus associated with CTEPH. This locus, however, consists only intronic SNPs and there is no mechanistic explanation on how these non-coding SNPs control the risk of CTEPH. Methods: GWAS-identified CTEPH-associated ABO locus was analyzed, and haplotype SNPs were identified based on linkage disequilibrium (LD). Electrophoretic mobility shift assay (EMSA) was applied to screen functional SNPs (fSNPs) based on allele-imbalanced nuclear protein binding. SNP-binding nuclear transcription factors (TFs) were identified by proteomics. Hi-C chromatin-interaction analysis was utilized to determine potential target genes affected by fSNPs. Knockdown of fSNP-associated TFs in human pulmonary artery endothelial cells (hPAECs) was applied to validate the regulation of TFs on target genes. Results: Based on GWAS, the minor allele of tag SNP rs2519093 is associated with higher risk of CTEPH (Odds ratio 2.22). It defines 11 haplotype SNPs in a LD with R 2 > 0.8. Screened by EMSA with hPAEC nuclear proteins, intronic SNPs rs579459 and rs550057 were identified functional given their allele-specific protein binding. Proteomics analysis identified 3 candidate TFs that allele-specifically bind to fSNP rs579459: PBX1, CTCF and SAFB2. Candidate target gene ADAMTS13 is chosen based on its prominent interaction with fSNP revealed by Hi-C analysis, and the potential role in thromboembolism through vWF cleavage. TF knockdown by siRNA downregulates ADAMTS13 in hPAECs, suggesting their regulatory function as activators. Conclusion: Leveraging our post-GWAS functional genomics approach, we identified fSNPs in ABO locus that define a genetic architecture controlling the susceptibility of CTEPH. This genetic architecture, along with the newly identified fSNP-associated TFs and target gene ADAMTS13, provides a biological explanation for the pathogenesis of CTEPH.
