Caiyun G. Li

FK506 activates BMPR2, rescues endothelial dysfunction, and reverses pulmonary hypertension

Dysfunctional bone morphogenetic protein receptor-2 (BMPR2) signaling is implicated in the pathogenesis of pulmonary arterial hypertension (PAH). We used a transcriptional high-throughput luciferase reporter assay to screen 3,756 FDA-approved drugs and bioactive compounds for induction of BMPR2 signaling. The best response was achieved with FK506 (tacrolimus), via a dual mechanism of action as a calcineurin inhibitor that also binds FK-binding protein-12 (FKBP12), a repressor of BMP signaling. FK506 released FKBP12 from type I receptors activin receptor-like kinase 1 (ALK1), ALK2, and ALK3 and activated downstream SMAD1/5 and MAPK signaling and ID1 gene regulation in a manner superior to the calcineurin inhibitor cyclosporine and the FKBP12 ligand rapamycin. In pulmonary artery endothelial cells (ECs) from patients with idiopathic PAH, low-dose FK506 reversed dysfunctional BMPR2 signaling. In mice with conditional Bmpr2 deletion in ECs, low-dose FK506 prevented exaggerated chronic hypoxic PAH associated with induction of EC targets of BMP signaling, such as apelin. Low-dose FK506 also reversed severe PAH in rats with medial hypertrophy following monocrotaline and in rats with neointima formation following VEGF receptor blockade and chronic hypoxia. Our studies indicate that low-dose FK506 could be useful in the treatment of PAH.

BMPR2 Preserves Mitochondrial Function and DNA during Reoxygenation to Promote Endothelial Cell Survival and Reverse Pulmonary Hypertension

Mitochondrial dysfunction, inflammation, and mutant bone morphogenetic protein receptor 2 (BMPR2) are associated with pulmonary arterial hypertension (PAH), an incurable disease characterized by pulmonary arterial (PA) endothelial cell (EC) apoptosis, decreased microvessels, and occlusive vascular remodeling. We hypothesized that reduced BMPR2 induces PAEC mitochondrial dysfunction, promoting a pro-inflammatory or pro-apoptotic state. Mice with EC deletion of BMPR2 develop hypoxia-induced pulmonary hypertension that, in contrast to non-transgenic littermates, does not reverse upon reoxygenation and is associated with reduced PA microvessels and lung EC p53, PGC1α and TFAM, regulators of mitochondrial biogenesis, and mitochondrial DNA. Decreasing PAEC BMPR2 by siRNA during reoxygenation represses p53, PGC1α, NRF2, TFAM, mitochondrial membrane potential, and ATP and induces mitochondrial DNA deletion and apoptosis. Reducing PAEC BMPR2 in normoxia increases p53, PGC1α, TFAM, mitochondrial membrane potential, ATP production, and glycolysis, and induces mitochondrial fission and a pro-inflammatory state. These features are recapitulated in PAECs from PAH patients with mutant BMPR2.

Elafin Reverses Pulmonary Hypertension via Caveolin-1–Dependent Bone Morphogenetic Protein Signaling

RATIONALE Pulmonary arterial hypertension is characterized by endothelial dysfunction, impaired bone morphogenetic protein receptor 2 (BMPR2) signaling, and increased elastase activity. Synthetic elastase inhibitors reverse experimental pulmonary hypertension but cause hepatotoxicity in clinical studies. The endogenous elastase inhibitor elafin attenuates hypoxic pulmonary hypertension in mice, but its potential to improve endothelial function and BMPR2 signaling, and to reverse severe experimental pulmonary hypertension or vascular pathology in the human disease was unknown. OBJECTIVES To assess elafin-mediated regression of pulmonary vascular pathology in rats and in lung explants from patients with pulmonary hypertension. To determine if elafin amplifies BMPR2 signaling in pulmonary artery endothelial cells and to elucidate the underlying mechanism. METHODS Rats with pulmonary hypertension induced by vascular endothelial growth factor receptor blockade and hypoxia (Sugen/hypoxia) as well as lung organ cultures from patients with pulmonary hypertension were used to assess elafin-mediated reversibility of pulmonary vascular disease. Pulmonary arterial endothelial cells from patients and control subjects were used to determine the efficacy and mechanism of elafin-mediated BMPR2 signaling. MEASUREMENTS AND MAIN RESULTS In Sugen/hypoxia rats, elafin reduced elastase activity and reversed pulmonary hypertension, judged by regression of right ventricular systolic pressure and hypertrophy and pulmonary artery occlusive changes. Elafin improved endothelial function by increasing apelin, a BMPR2 target. Elafin induced apoptosis in human pulmonary arterial smooth muscle cells and decreased neointimal lesions in lung organ culture. In normal and patient pulmonary artery endothelial cells, elafin promoted angiogenesis by increasing pSMAD-dependent and -independent BMPR2 signaling. This was linked mechanistically to augmented interaction of BMPR2 with caveolin-1 via elafin-mediated stabilization of endothelial surface caveolin-1. CONCLUSIONS Elafin reverses obliterative changes in pulmonary arteries via elastase inhibition and caveolin-1-dependent amplification of BMPR2 signaling.

PAX8 promotes tumor cell growth by transcriptionally regulating E2F1 and stabilizing RB protein

The retinoblastoma protein (RB)–E2F1 pathway has a central role in regulating the cell cycle. Several PAX proteins (tissue-specific developmental regulators), including PAX8, interact with the RB protein, and thus regulate the cell cycle directly or indirectly. Here, we report that PAX8 expression is frequent in renal cell carcinoma, bladder, ovarian and thyroid cancer cell lines, and that silencing of PAX8 in cancer cell lines leads to a striking reduction in the expression of E2F1 and its target genes, as well as a proteasome-dependent destabilization of RB protein, with the RB1 mRNA level remaining unaffected. Cancer cells expressing PAX8 undergo a G1/S arrest and eventually senesce following PAX8 silencing. We demonstrate that PAX8 transcriptionally regulates the E2F1 promoter directly, and E2F1 transcription is enhanced after RB depletion. RB is recruited to the PAX8-binding site, and is involved in PAX8-mediated E2F1 transcription in cancer cells. Therefore, our results suggest that, in cancer, frequent and persistent expression of PAX8 is required for cell growth control through transcriptional activation of E2F1 expression and upregulation of the RB–E2F1 pathway.

Whole-Exome Sequencing Reveals TopBP1 as a Novel Gene in Idiopathic Pulmonary Arterial Hypertension

RATIONALE Idiopathic pulmonary arterial hypertension (IPAH) is a life-threatening disorder characterized by progressive loss of pulmonary microvessels. Although mutations in the bone morphogenetic receptor 2 (BMPR2) are found in 80% of heritable and ∼15% of patients with IPAH, their low penetrance (∼20%) suggests that other unidentified genetic modifiers are required for manifestation of the disease phenotype. Use of whole-exome sequencing (WES) has recently led to the discovery of novel susceptibility genes in heritable PAH, but whether WES can also accelerate gene discovery in IPAH remains unknown. OBJECTIVES To determine whether WES can help identify novel gene modifiers in patients with IPAH. METHODS Exome capture and sequencing was performed on genomic DNA isolated from 12 unrelated patients with IPAH lacking BMPR2 mutations. Observed genetic variants were prioritized according to their pathogenic potential using ANNOVAR. MEASUREMENTS AND MAIN RESULTS A total of nine genes were identified as high-priority candidates. Our top hit was topoisomerase DNA binding II binding protein 1 (TopBP1), a gene involved in the response to DNA damage and replication stress. We found that TopBP1 expression was reduced in vascular lesions and pulmonary endothelial cells isolated from patients with IPAH. Although TopBP1 deficiency made endothelial cells susceptible to DNA damage and apoptosis in response to hydroxyurea, its restoration resulted in less DNA damage and improved cell survival. CONCLUSIONS WES led to the discovery of TopBP1, a gene whose deficiency may increase susceptibility to small vessel loss in IPAH. We predict that use of WES will help identify gene modifiers that influence an individuals risk of developing IPAH.

PAX Genes in Cancer; Friends or Foes?

PAX genes have been shown to be critically required for the development of specific tissues and organs during embryogenesis. In addition, PAX genes are expressed in a handful of adult tissues where they are thought to play important roles, usually different from those in embryogenesis. A common theme in adult tissues is a requirement for PAX gene expression in adult stem cell maintenance or tissue regeneration. The connections between adult stem cell PAX gene expression and cancer are intriguing, and the literature is replete with examples of PAX gene expression in either situation. Here we systematically review the literature and present an overview of postnatal PAX gene expression in normal and cancerous tissue. We discuss the potential link between PAX gene expression in adult tissue and cancer. In addition, we discuss whether persistent PAX gene expression in cancer is favorable or unfavorable.

RNA Sequencing Analysis Detection of a Novel Pathway of Endothelial Dysfunction in Pulmonary Arterial Hypertension.

RATIONALE Pulmonary arterial hypertension is characterized by endothelial dysregulation, but global changes in gene expression have not been related to perturbations in function. OBJECTIVES RNA sequencing was used to discriminate changes in transcriptomes of endothelial cells cultured from lungs of patients with idiopathic pulmonary arterial hypertension versus control subjects and to assess the functional significance of major differentially expressed transcripts. METHODS The endothelial transcriptomes from the lungs of seven control subjects and six patients with idiopathic pulmonary arterial hypertension were analyzed. Differentially expressed genes were related to bone morphogenetic protein type 2 receptor (BMPR2) signaling. Those down-regulated were assessed for function in cultured cells and in a transgenic mouse. MEASUREMENTS AND MAIN RESULTS Fold differences in 10 genes were significant (P < 0.05), four increased and six decreased in patients versus control subjects. No patient was mutant for BMPR2. However, knockdown of BMPR2 by siRNA in control pulmonary arterial endothelial cells recapitulated 6 of 10 patient-related gene changes, including decreased collagen IV (COL4A1, COL4A2) and ephrinA1 (EFNA1). Reduction of BMPR2-regulated transcripts was related to decreased β-catenin. Reducing COL4A1, COL4A2, and EFNA1 by siRNA inhibited pulmonary endothelial adhesion, migration, and tube formation. In mice null for the EFNA1 receptor, EphA2, versus control animals, vascular endothelial growth factor receptor blockade and hypoxia caused more severe pulmonary hypertension, judged by elevated right ventricular systolic pressure, right ventricular hypertrophy, and loss of small arteries. CONCLUSIONS The novel relationship between BMPR2 dysfunction and reduced expression of endothelial COL4 and EFNA1 may underlie vulnerability to injury in pulmonary arterial hypertension.

iPSC Model of Pulmonary Arterial Hypertension Reveals Novel Gene Expression and Patient Specificity.

Rationale: Idiopathic or heritable pulmonary arterial hypertension is characterized by loss and obliteration of lung vasculature. Endothelial cell dysfunction is pivotal to the pathophysiology, but different causal mechanisms may reflect a need for patient‐tailored therapies. Objectives: Endothelial cells differentiated from induced pluripotent stem cells were compared with pulmonary arterial endothelial cells from the same patients with idiopathic or heritable pulmonary arterial hypertension, to determine whether they shared functional abnormalities and altered gene expression patterns that differed from those in unused donor cells. We then investigated whether endothelial cells differentiated from pluripotent cells could serve as surrogates to test emerging therapies. Methods: Functional changes assessed included adhesion, migration, tube formation, and propensity to apoptosis. Expression of bone morphogenetic protein receptor type 2 (BMPR2) and its target, collagen IV, signaling of the phosphorylated form of the mothers against decapentaplegic proteins (pSMAD1/5), and transcriptomic profiles were also analyzed. Measurements and Main Results: Native pulmonary arterial and induced pluripotent stem cell‐derived endothelial cells from patients with idiopathic and heritable pulmonary arterial hypertension compared with control subjects showed a similar reduction in adhesion, migration, survival, and tube formation, and decreased BMPR2 and downstream signaling and collagen IV expression. Transcriptomic profiling revealed high kisspeptin 1 (KISS1) related to reduced migration and low carboxylesterase 1 (CES1), to impaired survival in patient cells. A beneficial angiogenic response to potential therapies, FK506 and Elafin, was related to reduced slit guidance ligand 3 (SLIT3), an antimigratory factor. Conclusions: Despite the site of disease in the lung, our study indicates that induced pluripotent stem cell‐derived endothelial cells are useful surrogates to uncover novel features related to disease mechanisms and to better match patients to therapies.

Loss of Adenomatous Poliposis Coli-α3 Integrin Interaction Promotes Endothelial Apoptosis in Mice and Humans

Rationale: Pulmonary hypertension (PH) is characterized by progressive elevation in pulmonary pressure and loss of small pulmonary arteries. As bone morphogenetic proteins promote pulmonary angiogenesis by recruiting the Wnt/&bgr;-catenin pathway, we proposed that &bgr;-catenin activation could reduce loss and induce regeneration of small pulmonary arteries (PAs) and attenuate PH. Objective: This study aims to establish the role of &bgr;-catenin in protecting the pulmonary endothelium and stimulating compensatory angiogenesis after injury. Methods and Results: To assess the impact of &bgr;-catenin activation on chronic hypoxia-induced PH, we used the adenomatous polyposis coli (ApcMin/+) mouse, where reduced APC causes constitutive &bgr;-catenin elevation. Surprisingly, hypoxic ApcMin/+ mice displayed greater PH and small PA loss compared with control C57Bl6J littermates. PA endothelial cells isolated from ApcMin/+ demonstrated reduced survival and angiogenic responses along with a profound reduction in adhesion to laminin. The mechanism involved failure of APC to interact with the cytoplasmic domain of the &agr;3 integrin, to stabilize focal adhesions and activate integrin-linked kinase-1 and phospho Akt. We found that PA endothelial cells from lungs of patients with idiopathic PH have reduced APC expression, decreased adhesion to laminin, and impaired vascular tube formation. These defects were corrected in the cultured cells by transfection of APC. Conclusions: We show that APC is integral to PA endothelial cells adhesion and survival and is reduced in PA endothelial cells from PH patient lungs. The data suggest that decreased APC may be a cause of increased risk or severity of PH in genetically susceptible individuals.

PAX3 knockdown in metastatic melanoma cell lines does not reduce MITF expression.

PAX3 and MITF are important transcriptional activators in the melanocyte lineage and PAX3 is thought to control MITF expression during normal melanocyte differentiation. However, it is not clear whether this is still true in melanoma and whether the effects of knockdown of PAX3 on the inhibition of melanoma growth or survival are by its regulation of MITF. By western blot and quantitative real-time reverse transcription-PCR, we investigated the relationship between PAX3 and MITF expression in 27 metastatic melanoma and one immortalized melanocyte cell lines. All lines were found to express both PAX3 and MITF proteins but levels varied by 15 fold and more than 100 fold, respectively. The expression of PAX3 protein was correlated with that of MITF (r=0.75; P<0.001) but the expression of PAX3 protein and MITF mRNA was not. Immunofluorescence microscopy showed that individual cells expressed widely differing relative amounts of PAX3 and MITF protein. By MTT cell proliferation and flow cytometry assays, both MITF and PAX3 proteins seemed to be functional, as knockdown with siRNA led to reduced proliferation and induction of apoptosis. However, knockdown of PAX3 with small interfering RNA did not decrease MITF expression and vice versa. In one cell line (NZM15), silencing of PAX3 induced terminal differentiation whereas silencing of MITF induced expression of FOXD3, a repressor of melanogenesis. The results suggest that the melanoma lines used in this study show considerable phenotypic variation of expression of these two transcriptional activators and reflect a deregulation of the developmental process operating in the genesis of the melanocyte lineage, and that they probably function independently to enhance the survival of melanoma cells.