Proteomics- and Metabolomics-Based Analysis of Metabolic Changes in a Swine Model of Pulmonary Hypertension.

Pulmonary vein stenosis (PVS) causes a rare type of pulmonary hypertension (PH) by impacting the flow and pressure within the pulmonary vasculature, resulting in endothelial dysfunction and metabolic changes. A prudent line of treatment in this type of PH would be targeted therapy to relieve the pressure and reverse the flow-related changes. We used a swine model in order to mimic PH after PVS using pulmonary vein banding (PVB) of the lower lobes for 12 weeks to mimic the hemodynamic profile associated with PH and investigated the molecular alterations that provide an impetus for the development of PH. Our current study aimed to employ unbiased proteomic and metabolomic analyses on both the upper and lower lobes of the swine lung to identify regions with metabolic alterations. We detected changes in the upper lobes for the PVB animals mainly pertaining to fatty acid metabolism, reactive oxygen species (ROS) signaling and extracellular matrix (ECM) remodeling and small, albeit, significant changes in the lower lobes for purine metabolism.

Loss of lung microvascular endothelial Piezo2 expression impairs NO synthesis, induces EndMT, and is associated with pulmonary hypertension.

Mechanical forces are translated into biochemical stimuli by mechanotransduction channels, such as the mechanically activated cation channel Piezo2. Lung Piezo2 expression has recently been shown to be restricted to endothelial cells. Hence, we aimed to investigate the role of Piezo2 in regulation of pulmonary vascular function and structure, as well as its contribution to development of pulmonary arterial hypertension (PAH). The expression of Piezo2 was significantly reduced in pulmonary microvascular endothelial cells (MVECs) from patients with PAH, in lung tissue from mice with a Bmpr2+/R899X knock-in mutation commonly found in patients with pulmonary hypertension, and in lung tissue of monocrotaline (MCT) and sugen-hypoxia-induced PH (SuHx) PAH rat models, as well as from a swine model with pulmonary vein banding. In MVECs, Piezo2 expression was reduced in response to abnormal shear stress, hypoxia, and TGFß stimulation. Functional studies in MVECs exposed to shear stress illustrated that siRNA-mediated Piezo2 knockdown impaired endothelial alignment, calcium influx, phosphorylation of AKT, and nitric oxide production. In addition, siPiezo2 reduced the expression of the endothelial marker PECAM-1 and increased the expression of vascular smooth muscle markers ACTA2, SM22a, and calponin. Thus, Piezo2 acts as a mechanotransduction channel in pulmonary MVECs, stimulating shear-induced production of nitric oxide and is essentially involved in preventing endothelial to mesenchymal transition. Its blunted expression in pulmonary hypertension could impair the vasodilator capacity and stimulate vascular remodeling, indicating that Piezo2 might be an interesting therapeutic target to attenuate progression of the disease.NEW & NOTEWORTHY The mechanosensory ion channel Piezo2 is exclusively expressed in lung microvascular endothelial cells (MVECs). Patient MVECs as well as animal models of pulmonary (arterial) hypertension showed lower expression of Piezo2 in the lung. Mechanistically, Piezo2 is required for calcium influx and NO production in response to shear stress, whereas stimuli known to induce endothelial to mesenchymal transition (EndMT) reduce Piezo2 expression in MVECs, and Piezo2 knockdown induces a gene and protein expression pattern consistent with EndMT.

Cgnl1, an endothelial junction complex protein, regulates GTPase mediated angiogenesis.

AIMS: The formation of cell-cell and cell-extra cellular matrix (ECM) contacts by endothelial cells (ECs) is crucial for the stability and integrity of a vascular network. We previously identified cingulin-like 1 (Cgnl1) in a transcriptomic screen for new angiogenic modulators. Here we aim to study the function of the cell-cell junction associated protein Cgnl1 during vessel formation. METHODS AND RESULTS: Unlike family member cingulin, Cgnl1 expression is enriched in ECs during vascular growth. Cgnl1 is important for the formation of multicellular tubule structures, as shown in vitro using loss-of function assays in a 3D matrix co-culture system that uses primary human ECs and supporting mural cells. Further studies revealed that Cgnl1 regulates vascular growth by promoting Ve-cadherin association with the actin cytoskeleton, thereby stabilizing adherens junctions. Cgnl1 also regulates focal adhesion assembly in response to ECM contact, promoting vinculin and paxillin recruitment and focal adhesion kinase signalling. In vivo, we demonstrate in a postnatal retinal vascular development model in mice that Cgnl1 function is crucial for sustaining neovascular growth and stability. CONCLUSIONS: Our data demonstrate a functional relevance for Cgnl1 as a defining factor in new vessel formation both in vitro and in vivo.

Time course of VCAM-1 expression in reperfused myocardial infarction in swine and its relation to retention of intracoronary administered bone marrow-derived mononuclear cells.

BACKGROUND: Intracoronary infusion of autologous bone marrow-derived mononuclear cells (BMMNC), after acute myocardial infarction (AMI), has been shown to improve myocardial function. However, therapeutic efficacy is limited, possibly because cell retention rates are low, suggesting that optimization of cell retention might increase therapeutic efficacy. Since retention of injected BMMNC is observed only within infarcted, but not remote, myocardium, we hypothesized that adhesion molecules on activated endothelium following reperfusion are essential. Consequently, we investigated the role of vascular cell adhesion molecule 1 (VCAM-1) in BMMNC retention in swine undergoing reperfused AMI produced by 120 min of percutaneous left circumflex coronary occlusion. METHODS AND RESULTS: VCAM-1 expression in the infarct and remote region was quantified at 1, 3, 7, 14, and 35 days, post-reperfusion (n≥6 swine per group). Since expression levels were significantly higher at 3 days (2.41±0.62%) than at 7 days (0.98±0.28%; p<0.05), we compared the degree of cell retention at those time points in a follow-up study, in which an average of 43·106 autologous BMMNCs were infused intracoronary at 3, or 7 days, post-reperfusion (n = 6 swine per group) and retention was histologically quantified one hour after intracoronary infusion of autologous BMMNCs. Although VCAM-1 expression correlated with retention of BMMNC within each time point, overall BMMNC retention was similar at day 3 and day 7 (2.3±1.3% vs. 3.1±1.4%, p = 0.72). This was not due to the composition of infused bone marrow cell fractions (analyzed with flow cytometry; n = 5 per group), as cell composition of the infused BMMNC fractions was similar. CONCLUSION: These findings suggest that VCAM-1 expression influences to a small degree, but is not the principal determinant of, BMMNC retention.

THSD1 preserves vascular integrity and protects against intraplaque haemorrhaging in ApoE-/- mice.

AIMS: Impairment of the endothelial barrier leads to microvascular breakdown in cardiovascular disease and is involved in intraplaque haemorrhaging and the progression of advanced atherosclerotic lesions that are vulnerable to rupture. The exact mechanism that regulates vascular integrity requires further definition. Using a microarray screen for angiogenesis-associated genes during murine embryogenesis, we identified thrombospondin type I domain 1 (THSD1) as a new putative angiopotent factor with unknown biological function. We sought to characterize the role of THSD1 in endothelial cells during vascular development and cardiovascular disease. METHODS AND RESULTS: Functional knockdown of Thsd1 in zebrafish embryos and in a murine retina vascularization model induced severe haemorrhaging without affecting neovascular growth. In human carotid endarterectomy specimens, THSD1 expression by endothelial cells was detected in advanced atherosclerotic lesions with intraplaque haemorrhaging, but was absent in stable lesions, implying involvement of THSD1 in neovascular bleeding. In vitro, stimulation with pro-atherogenic factors (3% O2 and TNFα) decreased THSD1 expression in human endothelial cells, whereas stimulation with an anti-atherogenic factor (IL10) showed opposite effect. Therapeutic evaluation in a murine advanced atherosclerosis model showed that Thsd1 overexpression decreased plaque vulnerability by attenuating intraplaque vascular leakage, subsequently reducing macrophage accumulation and necrotic core size. Mechanistic studies in human endothelial cells demonstrated that THSD1 activates FAK-PI3K, leading to Rac1-mediated actin cytoskeleton regulation of adherens junctions and focal adhesion assembly. CONCLUSION: THSD1 is a new regulator of endothelial barrier function during vascular development and protects intraplaque microvessels against haemorrhaging in advanced atherosclerotic lesions.

Feasibility of intracoronary GLP-1 eluting CellBead™ infusion in acute myocardial infarction.

Cell therapy is a field of growing interest in the prevention of post acute myocardial infarction (AMI) heart failure. Stem cell retention upon local delivery to the heart, however, is still unsatisfactory. CellBeads were recently developed as a potential solution to this problem. CellBeads are 170-µm alginate microspheres that contain mesenchymal stem cells (MSCs) genetically modified to express glucagon-like peptide-1 (GLP-1) supplementary to inherent paracrine factors. GLP-1 is an incretin hormone that has both antiapoptotic and cardioprotective effects. Transplanting CellBeads in the post-AMI heart might induce cardiomyocyte salvage and ultimately abrogate adverse cardiac remodeling. We aimed to investigate the feasibility of intracoronary infusion of CellBeads in a large animal model of AMI. Four pigs were used in a pilot study to assess the maximal safe dose of CellBeads. In the remaining 21 animals, an AMI was induced by balloon occlusion of the left circumflex coronary artery for 90 min. During reperfusion, 60,000 CellBeads (n = 11), control beads (n = 4), or lactated Ringers' (n = 6) were infused. Animals were sacrificed after 2 or 7 days, and the hearts were excised for histological analyses. Intracoronary infusion did not permanently affect coronary flow in any of the groups. Histological analysis revealed CellBeads containing viable MSCs up to 7 days. Viability and activity of the MSCs was confirmed by qPCR analysis that showed expression of recombinant GLP-1 and human genes after 2 and 7 days. CellBeads reduced inflammatory infiltration by 29% (p = 0.001). In addition, they decreased the extent of apoptosis by 25% (p = 0.001) after 2 days. We show that intracoronary infusion of 5 million encapsulated MSCs is safe and feasible. Also, several parameters indicate that the cells have paracrine effects, suggesting a potential therapeutic benefit of this new approach.

Endothelial cell-specific FGD5 involvement in vascular pruning defines neovessel fate in mice.

BACKGROUND: New vessel formation contributes to organ development during embryogenesis and tissue repair in response to mechanical damage, inflammation, and ischemia in adult organisms. Early angiogenesis includes formation of an excessive primitive network that needs to be reorganized into a secondary vascular network with higher hierarchical structure. Vascular pruning, the removal of aberrant neovessels by apoptosis, is a vital step in this process. Although multiple molecular pathways for early angiogenesis have been identified, little is known about the genetic regulators of secondary network development. METHODS AND RESULTS: Using a transcriptomics approach, we identified a new endothelial specific gene named FYVE, RhoGEF, and PH domain-containing 5 (FGD5) that plays a crucial role in vascular pruning. Loss- and gain-of-function studies demonstrate that FGD5 inhibits neovascularization, indicated by in vitro tube-formation, aortic-ring, and coated-bead assays and by in vivo coated-bead plug assays and studies in the murine retina model. FGD5 promotes apoptosis-induced vaso-obliteration via induction of the hey1-p53 pathway by direct binding and activation of cdc42. Indeed, FGD5 correlates with apoptosis in endothelial cells during vascular remodeling and was linked to rising p21(CIP1) levels in aging mice. CONCLUSION: We have identified FGD5 as a novel genetic regulator of vascular pruning by activation of endothelial cell-targeted apoptosis.

PDGF-induced migration of vascular smooth muscle cells is inhibited by heme oxygenase-1 via VEGFR2 upregulation and subsequent assembly of inactive VEGFR2/PDGFRß heterodimers.

OBJECTIVE: In cardiovascular regulation, heme oxygenase-1 (HO-1) activity has been shown to inhibit vascular smooth muscle cell (VSMC) proliferation by promoting cell cycle arrest at the G1/S phase. However, the effect of HO-1 on VSMC migration remains unclear. We aim to elucidate the mechanism by which HO-1 regulates PDGFBB-induced VSMC migration. METHODS AND RESULTS: Transduction of HO-1 cDNA adenoviral vector severely impeded human VSMC migration in a scratch, transmembrane, and directional migration assay in response to PDGFBB stimulation. Similarly, HO-1 overexpression in the remodeling process during murine retinal vasculature development attenuated VSMC coverage over the major arterial branches as compared with sham vector-transduced eyes. HO-1 expression in VSMCs significantly upregulated VEGFA and VEGFR2 expression, which subsequently promoted the formation of inactive PDGFRß/VEGFR2 complexes. This compromised PDGFRß phosphorylation and impeded the downstream cascade of FAK-p38 signaling. siRNA-mediated silencing of VEGFA or VEGFR2 could reverse the inhibitory effect of HO-1 on VSMC migration. CONCLUSIONS: These findings identify a potent antimigratory function of HO-1 in VSMCs, a mechanism that involves VEGFA and VEGFR2 upregulation, followed by assembly of inactive VEGFR2/PDGFRß complexes that attenuates effective PDGFRß signaling.

Ets2 determines the inflammatory state of endothelial cells in advanced atherosclerotic lesions.

RATIONALE: Neovascularization is required for embryonic development and plays a central role in diseases in adults. In atherosclerosis, the role of neovascularization remains to be elucidated. In a genome-wide microarray-screen of Flk1+ angioblasts during murine embryogenesis, the v-ets erythroblastosis virus E26 oncogene homolog 2 (Ets2) transcription factor was identified as a potential angiogenic factor. OBJECTIVES: We assessed the role of Ets2 in endothelial cells during atherosclerotic lesion progression toward plaque instability. METHODS AND RESULTS: In 91 patients treated for carotid artery disease, Ets2 levels showed modest correlations with capillary growth, thrombogenicity, and rising levels of tumor necrosis factor-α (TNFα), monocyte chemoattractant protein 1, and interleukin-6 in the atherosclerotic lesions. Experiments in ApoE(-/-) mice, using a vulnerable plaque model, showed that Ets2 expression was increased under atherogenic conditions and was augmented specifically in the vulnerable versus stable lesions. In endothelial cell cultures, Ets2 expression and activation was responsive to the atherogenic cytokine TNFα. In the murine vulnerable plaque model, overexpression of Ets2 promoted lesion growth with neovessel formation, hemorrhaging, and plaque destabilization. In contrast, Ets2 silencing, using a lentiviral shRNA construct, promoted lesion stabilization. In vitro studies showed that Ets2 was crucial for TNFα-induced expression of monocyte chemoattractant protein 1, interleukin-6, and vascular cell adhesion molecule 1 in endothelial cells. In addition, Ets2 promoted tube formation and amplified TNFα-induced loss of vascular endothelial integrity. Evaluation in a murine retina model further validated the role of Ets2 in regulating vessel inflammation and endothelial leakage. CONCLUSIONS: We provide the first evidence for the plaque-destabilizing role of Ets2 in atherosclerosis development by induction of an intraplaque proinflammatory phenotype in endothelial cells.

Redundant roles for sox7 and sox18 in arteriovenous specification in zebrafish.

The specification of arteries and veins is an essential process in establishing and maintaining a functional blood vessel system. Incorrect arteriovenous specification disrupts embryonic development but has also been diagnosed in human syndromes such as hypotrichosis-lymphedema-telangiectasia, characterized by defects in blood and lymphatic vessels and associated with mutations in SOX18. Here we characterize the role of sox7 and sox18 during zebrafish vasculogenesis. Sox7 and sox18 are specifically expressed in the developing vasculature, and simultaneous loss of their function results in a severe loss of the arterial identity of the presumptive aorta which instead expresses venous markers, followed by dramatic arteriovenous shunt formations. Our study identifies members of the Sox family as key factors in specifying arteriovenous identity and will help to better understand hypotrichosis-lymphedema-telangiectasia and other diseases.

Measurement of antibodies to avian influenza virus A(H7N7) in humans by hemagglutination inhibition test.

During the epizootic of highly pathogenic avian influenza A(H7N7) in 2003 in The Netherlands, RT-PCR and culture confirmed infection was detected in 89 persons who were ill. A modified hemagglutination inhibition (HI) test using horse erythrocytes and 2 hemagglutinating units of virus was applied to assess retrospectively the extent of human (subclinical) infection. Validation of the HI-test with sera from 34 RT-PCR and culture confirmed A(H7) infected persons and sera from 100 persons from a human influenza vaccine trial in autumn 2002 showed that this HI-test had a sensitivity of 85% and a specificity of 100% when using a cut-off titer of > or =10. Using this cut-off value, A(H7) specific antibodies were detected in 49% of 508 persons exposed to poultry and in 64% of 63 persons exposed to A(H7) infected persons. Correlation of seropositivity with the occurrence of eye symptoms in exposed persons who had not received antiviral prophylaxis and of reduced seropositivity with taking antiviral prophylaxis provided further evidence that the A(H7) HI antibody titers were real. In conclusion, by applying an HI-test using horse erythrocytes human antibodies against the avian A(H7N7) virus were detected with high sensitivity and specificity in an unexpectedly high proportion of exposed persons.