NKX2-5 regulates vessel remodeling in scleroderma-associated pulmonary arterial hypertension.

NKX2-5 is a member of the homeobox-containing transcription factors critical in regulating tissue differentiation in development. Here, we report a role for NKX2-5 in vascular smooth muscle cell phenotypic modulation in vitro and in vascular remodeling in vivo. NKX2-5 is upregulated in scleroderma patients with pulmonary arterial hypertension. Suppression of NKX2-5 expression in smooth muscle cells halted vascular smooth muscle proliferation and migration, enhanced contractility, and blocked the expression of extracellular matrix genes. Conversely, overexpression of NKX2-5 suppressed the expression of contractile genes (ACTA2, TAGLN, CNN1) and enhanced the expression of matrix genes (COL1) in vascular smooth muscle cells. In vivo, conditional deletion of NKX2-5 attenuated blood vessel remodeling and halted the progression to hypertension in a mouse chronic hypoxia model. This study revealed that signals related to injury such as serum and low confluence, which induce NKX2-5 expression in cultured cells, is potentiated by TGF-ß and further enhanced by hypoxia. The effect of TGF-ß was sensitive to ERK5 and PI3K inhibition. Our data suggest a pivotal role for NKX2-5 in the phenotypic modulation of smooth muscle cells during pathological vascular remodeling and provide proof of concept for therapeutic targeting of NKX2-5 in vasculopathies.

Selective deletion of connective tissue growth factor attenuates experimentally-induced pulmonary fibrosis and pulmonary arterial hypertension.

Connective tissue growth factor (CTGF, CCN2) is a matricellular protein which plays key roles in normal mammalian development and in tissue homeostasis and repair. In pathological conditions, dysregulated CCN2 has been associated with cancer, cardiovascular disease, and tissue fibrosis. In this study, genetic manipulation of the CCN2 gene was employed to investigate the role of CCN2 expression in vitro and in experimentally-induced models of pulmonary fibrosis and pulmonary arterial hypertension (PAH). Knocking down CCN2 using siRNA reduced expression of pro-fibrotic markers (fibronectin p < 0.01, collagen type I p < 0.05, α-SMA p < 0.0001, TIMP-1 p < 0.05 and IL-6 p < 0.05) in TGF-ß-treated lung fibroblasts derived from systemic sclerosis patients. In vivo studies were performed in mice using a conditional gene deletion strategy targeting CCN2 in a fibroblast-specific and time-dependent manner in two models of lung disease. CCN2 deletion significantly reduced pulmonary interstitial scarring and fibrosis following bleomycin-instillation, as assessed by fibrotic scores (wildtype bleomycin 3.733 ± 0.2667 vs CCN2 knockout (KO) bleomycin 4.917 ± 0.3436, p < 0.05) and micro-CT. In the well-established chronic hypoxia/Sugen model of pulmonary hypertension, CCN2 gene deletion resulted in a significant decrease in pulmonary vessel remodelling, less right ventricular hypertrophy and a reduction in the haemodynamic measurements characteristic of PAH (RVSP and RV/LV + S were significantly reduced (p < 0.05) in CCN2 KO compared to WT mice in hypoxic/SU5416 conditions). These results support a prominent role for CCN2 in pulmonary fibrosis and in vessel remodelling associated with PAH. Therefore, therapeutics aimed at blocking CCN2 function are likely to benefit several forms of severe lung disease.

The Biochemical Literacy Framework: Inviting pedagogical innovation in higher education.

When developing meaningful curricula, institutions must engage with the desired disciplinary attributes of their graduates. Successfully employed in several areas, including psychology and chemistry, disciplinary literacies provide structure for the development of core competencies-pursuing progressive education. To this end, we have sought to develop a comprehensive blueprint of a graduate biochemist, providing detailed insight into the development of skills in the context of disciplinary knowledge. The Biochemical Literacy Framework (BCLF) aspires to encourage innovative course design in both the biochemical field and beyond through stimulating discussion among individuals developing undergraduate biochemistry degree courses based on pedagogical best practice. Here, we examine the concept of biochemical literacy aiming to start answering the question: What must individuals do and know to approach and transform ideas in the context of the biochemical sciences? The BCLF began with the guidance published by relevant learned societies - including the Royal Society of Biology, the Biochemical Society, the American Society for Biochemistry and Molecular Biology and the Quality Assurance Agency, before considering relevant pedagogical literature. We propose that biochemical literacy is comprised of seven key skills: critical thinking, self-management, communication, information literacy, visual literacy, practical skills and content knowledge. Together, these form a dynamic, highly interconnected and interrelated meta-literacy supporting the use of evidence-based, robust learning techniques. The BCLF is intended to form the foundation for discussion between colleagues, in addition to forming the groundwork for both pragmatic and exploratory future studies into facilitating and further defining biochemical literacy.

Endothelial to Mesenchymal Transition Contributes to Endothelial Dysfunction in Pulmonary Arterial Hypertension.

Pulmonary arterial hypertension (PAH) is a progressive disease characterized by lung endothelial cell dysfunction and vascular remodeling. Normally, the endothelium forms an integral cellular barrier to regulate vascular homeostasis. During embryogenesis endothelial cells exhibit substantial plasticity that contribute to cardiac development by undergoing endothelial-to-mesenchymal transition (EndoMT). We determined the presence of EndoMT in the pulmonary vasculature in vivo and the functional effects on pulmonary artery endothelial cells (PAECs) undergoing EndoMT in vitro. Histologic assessment of patients with systemic sclerosis-associated PAH and the hypoxia/SU5416 mouse model identified the presence von Willebrand factor/α-smooth muscle actin-positive endothelial cells in up to 5% of pulmonary vessels. Induced EndoMT in PAECs by inflammatory cytokines IL-1ß, tumor necrosis factor α, and transforming growth factor ß led to actin cytoskeleton reorganization and the development of a mesenchymal morphology. Induced EndoMT cells exhibited up-regulation of mesenchymal markers, including collagen type I and α-smooth muscle actin, and a reduction in endothelial cell and junctional proteins, including von Willebrand factor, CD31, occludin, and vascular endothelial-cadherin. Induced EndoMT monolayers failed to form viable biological barriers and induced enhanced leak in co-culture with PAECs. Induced EndoMT cells secreted significantly elevated proinflammatory cytokines, including IL-6, IL-8, and tumor necrosis factor α, and supported higher immune transendothelial migration compared with PAECs. These findings suggest that EndoMT may contribute to the development of PAH.

Impaired bone morphogenetic protein receptor II signaling in a transforming growth factor-ß-dependent mouse model of pulmonary hypertension and in systemic sclerosis.

RATIONALE: Up to 10% of patients with systemic sclerosis (SSc) develop pulmonary arterial hypertension (PAH). This risk persists throughout the disease and is time dependent, suggesting that SSc is a susceptibility factor. Outcome for SSc-PAH is poor compared with heritable or idiopathic forms, despite clinical and pathological similarities. Although susceptibility in heritable PAH and idiopathic PAH is strongly associated with gene mutations leading to reduced expression of bone morphogenetic protein receptor (BMPR) II, these mutations have not been observed in SSc-PAH. OBJECTIVES: To explore BMPRII expression and function in a mouse model of SSc (TßRIIΔk-fib) that is susceptible to developing pulmonary hypertension and in SSc lung. METHODS: BMPRII and downstream signaling pathways were profiled in lung tissue and fibroblasts from the TßRIIΔk-fib model, which develops pulmonary vasculopathy with pulmonary hypertension that is exacerbated by SU5416. Complementary studies examined SSc or control lung tissue and fibroblasts. MEASUREMENTS AND MAIN RESULTS: Our study shows reduced BMPRII, impaired signaling, and altered receptor turnover activity in a transforming growth factor (TGF)-ß-dependent mouse model of SSc-PAH. Similarly, a significant reduction in BMPRII expression is observed in SSc lung tissue and fibroblasts. Increased proteasomal degradation of BMPRII appears to underlie this and may result from heightened TGF-ß activity. CONCLUSIONS: We found reduced BMPRII protein in patients with SSc-PAH and a relevant mouse model associated with increased proteasomal degradation of BMPRII. Collectively, these results suggest that impaired BMP signaling, resulting from TGF-ß-dependent increased receptor degradation, may promote PAH susceptibility in SSc and provide a unifying mechanism across different forms of PAH.

Inhibition of phosphodiesterase 2 augments cGMP and cAMP signaling to ameliorate pulmonary hypertension.

BACKGROUND: Pulmonary hypertension (PH) is a life-threatening disorder characterized by increased pulmonary artery pressure, remodeling of the pulmonary vasculature, and right ventricular failure. Loss of endothelium-derived nitric oxide (NO) and prostacyclin contributes to PH pathogenesis, and current therapies are targeted to restore these pathways. Phosphodiesterases (PDEs) are a family of enzymes that break down cGMP and cAMP, which underpin the bioactivity of NO and prostacyclin. PDE5 inhibitors (eg, sildenafil) are licensed for PH, but a role for PDE2 in lung physiology and disease has yet to be established. Herein, we investigated whether PDE2 inhibition modulates pulmonary cyclic nucleotide signaling and ameliorates experimental PH. METHODS AND RESULTS: The selective PDE2 inhibitor BAY 60-7550 augmented atrial natriuretic peptide- and treprostinil-evoked pulmonary vascular relaxation in isolated arteries from chronically hypoxic rats. BAY 60-7550 prevented the onset of both hypoxia- and bleomycin-induced PH and produced a significantly greater reduction in disease severity when given in combination with a neutral endopeptidase inhibitor (enhances endogenous natriuretic peptides), trepostinil, inorganic nitrate (NO donor), or a PDE5 inhibitor. Proliferation of pulmonary artery smooth muscle cells from patients with pulmonary arterial hypertension was reduced by BAY 60-7550, an effect further enhanced in the presence of atrial natriuretic peptide, NO, and treprostinil. CONCLUSIONS: PDE2 inhibition elicits pulmonary dilation, prevents pulmonary vascular remodeling, and reduces the right ventricular hypertrophy characteristic of PH. This favorable pharmacodynamic profile is dependent on natriuretic peptide bioactivity and is additive with prostacyclin analogues, PDE5 inhibitor, and NO. PDE2 inhibition represents a viable, orally active therapy for PH.

Endothelial injury in a transforming growth factor ß-dependent mouse model of scleroderma induces pulmonary arterial hypertension.

OBJECTIVE: To delineate the constitutive pulmonary vascular phenotype of the TßRIIΔk-fib mouse model of scleroderma, and to selectively induce pulmonary endothelial cell injury using vascular endothelial growth factor (VEGF) inhibition to develop a model with features characteristic of pulmonary arterial hypertension (PAH). METHODS: The TßRIIΔk-fib mouse strain expresses a kinase-deficient transforming growth factor ß (TGFß) receptor type II driven by a fibroblast-specific promoter, leading to ligand-dependent up-regulation of TGFß signaling, and replicates key fibrotic features of scleroderma. Structural, biochemical, and functional assessments of pulmonary vessels, including in vivo hemodynamic studies, were performed before and following VEGF inhibition, which induced pulmonary endothelial cell apoptosis. These assessments included biochemical analysis of the TGFß and VEGF signaling axes in tissue sections and explanted smooth muscle cells. RESULTS: In the TßRIIΔk-fib mouse strain, a constitutive pulmonary vasculopathy with medial thickening, a perivascular proliferating chronic inflammatory cell infiltrate, and mildly elevated pulmonary artery pressure resembled the well-described chronic hypoxia model of pulmonary hypertension. Following administration of SU5416, the pulmonary vascular phenotype was more florid, with pulmonary arteriolar luminal obliteration by apoptosis-resistant proliferating endothelial cells. These changes resulted in right ventricular hypertrophy, confirming hemodynamically significant PAH. Altered expression of TGFß and VEGF ligand and receptor was consistent with a scleroderma phenotype. CONCLUSION: In this study, we replicated key features of systemic sclerosis-related PAH in a mouse model. Our results suggest that pulmonary endothelial cell injury in a genetically susceptible mouse strain triggers this complication and support the underlying role of functional interplay between TGFß and VEGF, which provides insight into the pathogenesis of this disease.

Dietary nitrate ameliorates pulmonary hypertension: cytoprotective role for endothelial nitric oxide synthase and xanthine oxidoreductase.

BACKGROUND: Pulmonary hypertension (PH) is a multifactorial disease characterized by increased pulmonary vascular resistance and right ventricular failure; morbidity and mortality remain unacceptably high. Loss of nitric oxide (NO) bioactivity is thought to contribute to the pathogenesis of PH, and agents that augment pulmonary NO signaling are clinically effective in the disease. Inorganic nitrate (NO(3)(-)) and nitrite (NO(2)(-)) elicit a reduction in systemic blood pressure in healthy individuals; this effect is underpinned by endogenous and sequential reduction to NO. Herein, we determined whether dietary nitrate and nitrite might be preferentially reduced to NO by the hypoxia associated with PH, and thereby offer a convenient, inexpensive method of supplementing NO functionality to reduce disease severity. METHODS AND RESULTS: Dietary nitrate reduced the right ventricular pressure and hypertrophy, and pulmonary vascular remodeling in wild-type mice exposed to 3 weeks of hypoxia; this beneficial activity was mirrored largely by dietary nitrite. The cytoprotective effects of dietary nitrate were associated with increased plasma and lung concentrations of nitrite and cGMP. The beneficial effects of dietary nitrate and nitrite were reduced in mice lacking endothelial NO synthase or treated with the xanthine oxidoreductase inhibitor allopurinol. CONCLUSIONS: These data demonstrate that dietary nitrate, and to a lesser extent dietary nitrite, elicit pulmonary dilatation, prevent pulmonary vascular remodeling, and reduce the right ventricular hypertrophy characteristic of PH. This favorable pharmacodynamic profile depends on endothelial NO synthase and xanthine oxidoreductase -catalyzed reduction of nitrite to NO. Exploitation of this mechanism (ie, dietary nitrate/nitrite supplementation) represents a viable, orally active therapy for PH.