Nicotine dose-dependent epigenomic-wide DNA methylation changes in the mice with long-term electronic cigarette exposure.

Epigenomic-wide DNA methylation profiling holds the potential to reflect both electronic cigarette exposure-associated risks and individual poor health outcomes. However, a systemic study in animals or humans is still lacking. Using the Infinium Mouse Methylation BeadChip, we examined the DNA methylation status of white blood cells in male ApoE-/- mice after 14 weeks of electronic cigarette exposure with the InExpose system (2 hr/day, 5 days/week, 50% PG and 50% VG) with low (6 mg/ml) and high (36 mg/ml) nicotine concentrations. Our results indicate that electronic cigarette aerosol inhalation induces significant alteration of 8,985 CpGs in a dose-dependent manner (FDR<0.05); 7,389 (82.2%) of the CpG sites are annotated with known genes. Among the top 6 significant CpG sites (P-value<1e-8), 4 CpG sites are located in the known genes, and most (3/5) of these genes have been related to cigarette smoking. The other two CpGs are close to/associated with the Phc2 gene that was recently linked to smoking in a transcriptome-wide associations study. Furthermore, the gene set enrichment analysis highlights the activation of MAPK and 4 cardiomyocyte/cardiomyopathy-related signaling pathways (including adrenergic signaling in cardiomyocytes and arrhythmogenic right ventricular cardiomyopathy) following repeated electronic cigarette use. The MAPK pathway activation correlates well with our finding of increased cytokine mRNA expression after electronic cigarette exposure in the same batch of mice. Interestingly, two pathways related to mitochondrial activities, namely mitochondrial gene expression and mitochondrial translation, are also activated after electronic cigarette exposure. Elucidating the relationship between these pathways and the increased circulating mitochondrial DNA observed here will provide further insight into the cell-damaging effects of prolonged inhalation of e-cigarette aerosols.

MEKK3-TGFß crosstalk regulates inward arterial remodeling.

Arterial remodeling is an important adaptive mechanism that maintains normal fluid shear stress in a variety of physiologic and pathologic conditions. Inward remodeling, a process that leads to reduction in arterial diameter, plays a critical role in progression of such common diseases as hypertension and atherosclerosis. Yet, despite its pathogenic importance, molecular mechanisms controlling inward remodeling remain undefined. Mitogen-activated protein kinases (MAPKs) perform a number of functions ranging from control of proliferation to migration and cell-fate transitions. While the MAPK ERK1/2 signaling pathway has been extensively examined in the endothelium, less is known about the role of the MEKK3/ERK5 pathway in vascular remodeling. To better define the role played by this signaling cascade, we studied the effect of endothelial-specific deletion of its key upstream MAP3K, MEKK3, in adult mice. The gene's deletion resulted in a gradual inward remodeling of both pulmonary and systematic arteries, leading to spontaneous hypertension in both vascular circuits and accelerated progression of atherosclerosis in hyperlipidemic mice. Molecular analysis revealed activation of TGFß-signaling both in vitro and in vivo. Endothelial-specific TGFßR1 knockout prevented inward arterial remodeling in MEKK3 endothelial knockout mice. These data point to the unexpected participation of endothelial MEKK3 in regulation of TGFßR1-Smad2/3 signaling and inward arterial remodeling in artery diseases.

Isoform-Specific Roles of ERK1 and ERK2 in Arteriogenesis.

Despite the clinical importance of arteriogenesis, this biological process is poorly understood. ERK1 and ERK2 are key components of a major intracellular signaling pathway activated by vascular endothelial growth (VEGF) and FGF2, growth factors critical to arteriogenesis. To investigate the specific role of each ERK isoform in arteriogenesis, we used mice with a global Erk1 knockout as well as Erk1 and Erk2 floxed mice to delete Erk1 or Erk2 in endothelial cells, macrophages, and smooth muscle cells. We found that ERK1 controls macrophage infiltration following an ischemic event. Loss of ERK1 in endothelial cells and macrophages induced an excessive macrophage infiltration leading to an increased but poorly functional arteriogenesis. Loss of ERK2 in endothelial cells leads to a decreased arteriogenesis due to decreased endothelial cell proliferation and a reduced eNOS expression. These findings show for the first time that isoform-specific roles of ERK1 and ERK2 in the control of arteriogenesis.

Noninvasive In Vivo Quantification of Adeno-Associated Virus Serotype 9-Mediated Expression of the Sodium/Iodide Symporter Under Hindlimb Ischemia and Neuraminidase Desialylation in Skeletal Muscle Using Single-Photon Emission Computed Tomography/Computed Tomography.

BACKGROUND: We propose micro single-photon emission computed tomography/computed tomography imaging of the hNIS (human sodium/iodide symporter) to noninvasively quantify adeno-associated virus 9 (AAV9)-mediated gene expression in a murine model of peripheral artery disease. METHODS: AAV9-hNIS (2×1011 viral genome particles) was injected into nonischemic or ischemic gastrocnemius muscles of C57Bl/6J mice following unilateral hindlimb ischemia ± the α-sialidase NA (neuraminidase). Control nonischemic limbs were injected with phosphate buffered saline or remained noninjected. Twelve mice underwent micro single-photon emission computed tomography/computed tomography imaging after serial injection of pertechnetate (99mTcO4-), a NIS substrate, up to 28 days after AAV9-hNIS injection. Twenty four animals were euthanized at selected times over 1 month for ex vivo validation. Forty-two animals were imaged with 99mTcO4- ± the selective NIS inhibitor perchlorate on day 10, to ascertain specificity of radiotracer uptake. Tissue was harvested for ex vivo validation. A modified version of the U-Net deep learning algorithm was used for image quantification. RESULTS: As quantitated by standardized uptake value, there was a gradual temporal increase in 99mTcO4- uptake in muscles treated with AAV9-hNIS. Hindlimb ischemia, NA, and hindlimb ischemia plus NA increased the magnitude of 99mTcO4- uptake by 4- to 5-fold compared with nonischemic muscle treated with only AAV9-hNIS. Perchlorate treatment significantly reduced 99mTcO4- uptake in AAV9-hNIS-treated muscles, demonstrating uptake specificity. The imaging results correlated well with ex vivo well counting (r2=0.9375; P<0.0001) and immunoblot analysis of NIS protein (r2=0.65; P<0.0001). CONCLUSIONS: Micro single-photon emission computed tomography/computed tomography imaging of hNIS-mediated 99mTcO4- uptake allows for accurate in vivo quantification of AAV9-driven gene expression, which increases under ischemic conditions or neuraminidase desialylation in skeletal muscle.

N-terminal syndecan-2 domain selectively enhances 6-O heparan sulfate chains sulfation and promotes VEGFA165-dependent neovascularization.

The proteoglycan Syndecan-2 (Sdc2) has been implicated in regulation of cytoskeleton organization, integrin signaling and developmental angiogenesis in zebrafish. Here we report that mice with global and inducible endothelial-specific deletion of Sdc2 display marked angiogenic and arteriogenic defects and impaired VEGFA165 signaling. No such abnormalities are observed in mice with deletion of the closely related Syndecan-4 (Sdc4) gene. These differences are due to a significantly higher 6-O sulfation level in Sdc2 versus Sdc4 heparan sulfate (HS) chains, leading to an increase in VEGFA165 binding sites and formation of a ternary Sdc2-VEGFA165-VEGFR2 complex which enhances VEGFR2 activation. The increased Sdc2 HS chains 6-O sulfation is driven by a specific N-terminal domain sequence; the insertion of this sequence in Sdc4 N-terminal domain increases 6-O sulfation of its HS chains and promotes Sdc2-VEGFA165-VEGFR2 complex formation. This demonstrates the existence of core protein-determined HS sulfation patterns that regulate specific biological activities.

Daily Lisinopril vs Placebo for Prevention of Chemoradiation-Induced Pulmonary Distress in Patients With Lung Cancer (Alliance MC1221): A Pilot Double-Blind Randomized Trial.

PURPOSE: Chemoradiation (CRT) is an integral treatment modality for patients with locally advanced lung cancer. It has been hypothesized that current use of an angiotensin-converting enzyme inhibitor during CRT may be protective for treatment-related lung damage and pneumonitis. METHODS AND MATERIALS: We conducted a pilot, double-blind, placebo-controlled, randomized trial. Study-eligible patients receiving curative thoracic radiation therapy (RT) were randomly assigned to 20 mg of lisinopril or placebo once daily during and up to 3 months after RT. All patients received concurrent chemotherapy. The primary endpoint was adverse event profiling. Multiple patient-reported outcome (PRO) surveys, including the Lung Cancer Symptom Scale, Function Assessment of Cancer Therapy-Lung, and the European Organisation for Research and Treatment of Cancer Lung Cancer Questionnaire, were applied with a symptom experience questionnaire. Exploratory comparative statistics were used to detect differences between arms with χ2 and Kruskal-Wallis testing. RESULTS: Five institutions enrolled 23 patients. However, accrual was less than expected. Eleven and 12 patients were in the placebo and lisinopril arms, respectively (mean age, 63.5 years; male, 62%). Baseline characteristics were balanced. Eighteen patients (86%) were former or current smokers. The primary endpoint was met; neither arm had grade 3 or higher hypotension, acute kidney injury, allergic reaction (medication-induced cough), or anaphylaxis (medication-related angioedema). Few PRO measures suggested that compared with the placebo arm, patients receiving lisinopril had less cough, less shortness of breath, fewer symptoms from lung cancer, less dyspnea with both walking and climbing stairs, and better overall quality of life (for all, P < .05). CONCLUSIONS: Although underpowered because of low accrual, our results suggest that there was a clinical signal for safety-and possibly beneficial by limited PRO measures-in concurrently administering lisinopril during thoracic CRT to mitigate or prevent RT-induced pulmonary distress. Our results showed that a definitive, larger-scale, randomized phase 3 trial is needed in the future.

[Lymphomatoid gastropathy: one case report and literatures review].

Objective: To report the first case of lymphomatoid gastropathy in China, and to demonstrate the clinical characteristics, diagnostic approach, treatment and prognosis in this kind of patients. Methods: One patient was diagnosed as lymphomatoid gastropathy at Peking Union Medical College Hospital, and her clinical characteristics, lab data, treatment and follow-up outcomes were reviewed. Results: A case of a 51-year-old female was presented, who underwent esophagogastroduodenoscopy (EGD) due to slight epigastric discomfort. EGD revealed multiple ulcers and erosions. Biopsies showed atypical lymphocytes infiltration with CD3(+), CD56(+), CD20(-), CD8(-), TIA(+), Granzyme B(-) and Ki-67 (75%). Epstein-Barr virus-encoded RNA in situ hybridization was negative. Four months later, repeated EGD examination showed regression of the lesions without specific treatment. Conclusion: Lymphomatoid gastropathy was a unique disease entity mimicking NK/T-cell lymphomas in pathology, with the quite different profile of treatment and prognosis. It's important to consider this issue during the differential diagnosis to avoid any excessive treatment.

SUMOylation of VEGFR2 regulates its intracellular trafficking and pathological angiogenesis.

Regulation of VEGFR2 represents an important mechanism for the control of angiogenesis. VEGFR2 activity can be regulated by post-translational modifications such as ubiquitination and acetylation. However, whether VEGFR2 can be regulated by SUMOylation has not been investigated. Here we show that endothelial-specific deletion of the SUMO endopeptidase SENP1 reduces pathological angiogenesis and tissue repair during hindlimb ischemia, and VEGF-induced angiogenesis in the cornea, retina, and ear. SENP1-deficient endothelial cells show increased SUMOylation of VEGFR2 and impaired VEGFR2 signalling. SUMOylation at lysine 1270 retains VEGFR2 in the Golgi and reduces its surface expression, attenuating VEGFR2-dependent signalling. Moreover, we find that SENP1 is downregulated and VEGFR2 hyper-SUMOylated in diabetic settings and that expression of a non-SUMOylated form of VEGFR2 rescues angiogenic defects in diabetic mice. These results show that VEGFR2 is regulated by deSUMOylation during pathological angiogenesis, and propose SENP1 as a potential therapeutic target for the treatment of diabetes-associated angiogenesis.

Inhibiting Integrin α5 Cytoplasmic Domain Signaling Reduces Atherosclerosis and Promotes Arteriogenesis.

BACKGROUND: Fibronectin in endothelial basement membranes promotes endothelial inflammatory activation and atherosclerosis but also promotes plaque stability and vascular remodeling. The fibronectin receptor α5 subunit is proinflammatory through binding to and activating phosphodiesterase 4D5, which inhibits anti-inflammatory cyclic adenosine monophosphate and protein kinase A. Replacing the α5 cytoplasmic domain with that of α2 resulted in smaller atherosclerotic plaques. Here, we further assessed plaque phenotype and compensatory vascular remodeling in this model. METHODS AND RESULTS: α5/2 mice in the hyperlipidemic apolipoprotein E null background had smaller plaques in the aortic root, with reduced endothelial NF-κB activation and inflammatory gene expression, reduced leukocyte content, and much lower metalloproteinase expression. However, smooth muscle cell content, fibrous cap thickness, and fibrillar collagen were unchanged, indicating no shift toward vulnerability. In vivo knockdown of phosphodiesterase 4D5 also decreased endothelial inflammatory activation and atherosclerotic plaque size. α5/2 mice showed improved recovery from hindlimb ischemia after femoral artery ligation. CONCLUSIONS: Blocking the fibronectin-Integrin α5 pathway reduces atherosclerotic plaque size, maintains plaque stability, and improves compensatory remodeling. This pathway is therefore a potential therapeutic target for treatment of atherosclerosis.

The effect of icariin on bone metabolism and its potential clinical application.

Osteoporosis is a bone disease characterized by reduced bone mass, which leads to increased risk of bone fractures, and poses a significant risk to public health, especially in the elderly population. The traditional Chinese medicinal herb Epimedii has been utilized for centuries to treat bone fracture and bone loss. Icariin is a prenylated flavonol glycoside isolated from Epimedium herb, and has been shown to be the main bioactive component. This review provides a comprehensive survey of previous studies on icariin, including its structure and function, effect on bone metabolism, and potential for clinical application. These studies show that icariin promotes bone formation by stimulating osteogenic differentiation of BMSCs (bone marrow-derived mesenchymal stem cells), while inhibiting osteoclastogenic differentiation and the bone resorption activity of osteoclasts. Furthermore, icariin has been shown to be more potent than other flavonoid compounds in promoting osteogenic differentiation and maturation of osteoblasts. A 24-month randomized double-blind placebo-controlled clinical trial reported that icariin was effective in preventing postmenopausal osteoporosis with relatively low side effects. In conclusion, icariin may represent a class of flavonoids with bone-promoting activity, which could be used as potential treatment of postmenopausal osteoporosis.

Hyperoxia causes miR-34a-mediated injury via angiopoietin-1 in neonatal lungs.

Hyperoxia-induced acute lung injury (HALI) is a key contributor to the pathogenesis of bronchopulmonary dysplasia (BPD) in neonates, for which no specific preventive or therapeutic agent is available. Here we show that lung micro-RNA (miR)-34a levels are significantly increased in lungs of neonatal mice exposed to hyperoxia. Deletion or inhibition of miR-34a improves the pulmonary phenotype and BPD-associated pulmonary arterial hypertension (PAH) in BPD mouse models, which, conversely, is worsened by miR-34a overexpression. Administration of angiopoietin-1, which is one of the downstream targets of miR34a, is able to ameliorate the BPD pulmonary and PAH phenotypes. Using three independent cohorts of human samples, we show that miR-34a expression is increased in type 2 alveolar epithelial cells in neonates with respiratory distress syndrome and BPD. Our data suggest that pharmacologic miR-34a inhibition may be a therapeutic option to prevent or ameliorate HALI/BPD in neonates.

Development of small diameter nanofiber tissue engineered arterial grafts.

The surgical repair of heart and vascular disease often requires implanting synthetic grafts. While synthetic grafts have been successfully used for medium-to-large sized arteries, applications for small diameter arteries (<6 mm) is limited due to high rates of occlusion by thrombosis. Our objective was to develop a tissue engineered vascular graft (TEVG) for small diameter arteries. TEVGs composed of polylactic acid nanofibers with inner luminal diameter between 0.5 and 0.6 mm were surgically implanted as infra-renal aortic interposition conduits in 25 female C17SCID/bg mice. Twelve mice were given sham operations. Survival of mice with TEVG grafts was 91.6% at 12 months post-implantation (sham group: 83.3%). No instances of graft stenosis or aneurysmal dilatation were observed over 12 months post-implantation, assessed by Doppler ultrasound and microCT. Histologic analysis of explanted TEVG grafts showed presence of CD31-positive endothelial monolayer and F4/80-positive macrophages after 4, 8, and 12 months in vivo. Cells positive for α-smooth muscle actin were observed within TEVG, demonstrating presence of smooth muscle cells (SMCs). Neo-extracellular matrix consisting mostly of collagen types I and III were observed at 12 months post-implantation. PCR analysis supports histological observations. TEVG group showed significant increases in expressions of SMC marker, collagen-I and III, matrix metalloproteinases-2 and 9, and itgam (a macrophage marker), when compared to sham group. Overall, patency rates were excellent at 12 months after implantation, as structural integrity of these TEVG. Tissue analysis also demonstrated vessel remodeling by autologous cell.

ELAVL1 regulates alternative splicing of eIF4E transporter to promote postnatal angiogenesis.

Posttranscriptional RNA regulation is important in determining the plasticity of cellular phenotypes. However, mechanisms of how RNA binding proteins (RBPs) influence cellular behavior are poorly understood. We show here that the RBP embryonic lethal abnormal vision like 1 (ELAVL1, also know as HuR) regulates the alternative splicing of eukaryotic translation initiation factor 4E nuclear import factor 1 (Eif4enif1), which encodes an eukaryotic translation initiation factor 4E transporter (4E-T) protein and suppresses the expression of capped mRNAs. In the absence of ELAVL1, skipping of exon 11 of Eif4enif1 forms the stable, short isoform, 4E-Ts. This alternative splicing event results in the formation of RNA processing bodies (PBs), enhanced turnover of angiogenic mRNAs, and suppressed sprouting behavior of vascular endothelial cells. Further, endothelial-specific Elavl1 knockout mice exhibited reduced revascularization after hind limb ischemia and tumor angiogenesis in oncogene-induced mammary cancer, resulting in attenuated blood flow and tumor growth, respectively. ELAVL1-regulated alternative splicing of Eif4enif1 leading to enhanced formation of PB and mRNA turnover constitutes a novel posttranscriptional mechanism critical for pathological angiogenesis.

A new variable for SRS plan quality evaluation based on normal tissue sparing: the effect of prescription isodose levels.

OBJECTIVE: A new dosimetric variable, dose-dropping speed (DDS), was proposed and used to evaluate normal tissue sparing among stereotactic radiosurgery (SRS) plans with different prescription isodose lines. METHODS: 40 plans were generated for 8 intracranial SRS cases, prescribing to isodose levels (IDLs) ranging from 50% to 90% in 10% increments. Whilst maintaining similar coverage and conformity, plans at different IDLs were evaluated in terms of normal tissue sparing using the proposed DDS. The DDS was defined as the greater decay coefficient in a double exponential decay fit of the dose drop-off outside the planning target volume (PTV), which models the steep portion of the drop-off. Provided that the prescription dose covers the whole PTV, a greater DDS indicates better normal tissue sparing. RESULTS: Among all plans, the DDS was found to be the lowest for the prescription at 90% IDL and the highest for the prescription at 60% or 70%. The beam profile slope change in the penumbra and its field size dependence were explored and given as the physical basis of the findings. CONCLUSION: A variable was proposed for SRS plan quality evaluation. Using this measure, prescriptions at 60% and 70% IDLs were found to provide best normal tissue sparing. ADVANCES IN KNOWLEDGE: A new variable was proposed based on which normal tissue sparing was quantitatively evaluated, comparing different prescription IDLs in SRS.

Chemokine-coupled ß2 integrin-induced macrophage Rac2-Myosin IIA interaction regulates VEGF-A mRNA stability and arteriogenesis.

Myeloid cells are important contributors to arteriogenesis, but their key molecular triggers and cellular effectors are largely unknown. We report, in inflammatory monocytes, that the combination of chemokine receptor (CCR2) and adhesion receptor (ß2 integrin) engagement leads to an interaction between activated Rac2 and Myosin 9 (Myh9), the heavy chain of Myosin IIA, resulting in augmented vascular endothelial growth factor A (VEGF-A) expression and induction of arteriogenesis. In human monocytes, CCL2 stimulation coupled to ICAM-1 adhesion led to rapid nuclear-to-cytosolic translocation of the RNA-binding protein HuR. This activation of HuR and its stabilization of VEGF-A mRNA were Rac2-dependent, and proteomic analysis for Rac2 interactors identified the 226 kD protein Myh9. The level of induced Rac2-Myh9 interaction strongly correlated with the degree of HuR translocation. CCL2-coupled ICAM-1 adhesion-driven HuR translocation and consequent VEGF-A mRNA stabilization were absent in Myh9(-/-) macrophages. Macrophage VEGF-A production, ischemic tissue VEGF-A levels, and flow recovery to hind limb ischemia were impaired in myeloid-specific Myh9(-/-) mice, despite preserved macrophage recruitment to the ischemic muscle. Micro-CT arteriography determined the impairment to be defective induced arteriogenesis, whereas developmental vasculogenesis was unaffected. These results place the macrophage at the center of ischemia-induced arteriogenesis, and they establish a novel role for Myosin IIA in signal transduction events modulating VEGF-A expression in tissue.

PTP1b is a physiologic regulator of vascular endothelial growth factor signaling in endothelial cells.

BACKGROUND: Regulation of vascular endothelial growth factor receptor-2 (VEGFR2) signaling is a control point that determines the extent of vascular tree formation. Recent studies demonstrated an important role played by VEGFR2 endothelial trafficking in control of its activity and suggested the involvement of a phosphotyrosine phosphatase 1b (PTP1b) in this process. This study was designed to define the role of PTP1b in endothelial VEGFR2 signaling and its role in regulation of angiogenesis and arteriogenesis. METHODS AND RESULTS: We generated mice carrying an endothelial-specific deletion of PTP1b and examined the effect of this knockout on VEGF signaling, angiogenesis, and arteriogenesis in vitro and in vivo. PTP1b knockout endothelial cells had increased VEGF-dependent activation of extracellular signal-regulated kinase signaling, sprouting, migration, and proliferation compared with controls. Endothelial PTP1b null mice had increased retinal and Matrigel implant angiogenesis and accelerated wound healing, pointing to enhanced angiogenesis. Increased arteriogenesis was demonstrated by observations of faster recovery of arterial blood flow and large numbers of newly formed arterioles in the hindlimb ischemia mouse model. PTP1b endothelial knockout also rescued impaired blood flow recovery after common femoral artery ligation in synectin null mice. CONCLUSIONS: PTP1b is a key regulator of endothelial VEGFR2 signaling and plays an important role in regulation of the extent of vascular tree formation.

Evaluation of cranial bone transport distraction with and without adipose grafting.

Transport distraction osteogenesis (DO) can be used to autologously reconstitute calvarial defects. The purpose of this study is to histomorphologically interrogate osteogenic formation during cranial transport distraction using a novel device. We also evaluate the effect of fat grafting on the regenerate and soft-tissue stability during distraction. This study was approved by Yale IACUC. Ten male New Zealand white rabbits (3 mo; 3.5 kg) were used (8 treatment, 2 control). A 16 × 16 mm defect was created abutted by a 10 × 16 mm transport disc. The device was fixated anterioposteriorly. Four animals were fat-grafted using 2 mL of subdermal intrascapular fat deposited along the distraction site. Latency (1 d), active distraction (12-14 d) (1.5 mm/d), and consolidation (4 wk) followed. Calcein and xylene orange fluorochromes were injected subcutaneously during and post-distraction to mark sites of bone formation. Following sacrifice, osteogenesis was assessed using microCT, histology, and fluorescence. Treatment animals demonstrated regenerate bone between distracted segments on microCT. MicroCT analysis of non-fat-grafted and fat-grafted animals revealed a mean density of 2271.95 mgHA/ccm and 2254.27 mgHA/ccm (P = 0.967), respectively, and defect bone versus total volume (BV/TV) of 0.0999 and 0.0766 (P = 0.5979), respectively. Controls had minimal reossification. Histologically, mean densities measured 43.63% and 8.19%, respectively. Fluorescence revealed ossification from the callus as well as from dura and periosteum in the cranial defect. Transport distraction is effective to reconstruct critically sized rabbit calvarial defects. Regenerate bone arises predominantly from the callus with contribution from surrounding dura and periosteum. Adipose grafting is well tolerated but does not enhance osseous regeneration.

VEGF-B-induced vascular growth leads to metabolic reprogramming and ischemia resistance in the heart.

Angiogenic growth factors have recently been linked to tissue metabolism. We have used genetic gain- and loss-of function models to elucidate the effects and mechanisms of action of vascular endothelial growth factor-B (VEGF-B) in the heart. A cardiomyocyte-specific VEGF-B transgene induced an expanded coronary arterial tree and reprogramming of cardiomyocyte metabolism. This was associated with protection against myocardial infarction and preservation of mitochondrial complex I function upon ischemia-reperfusion. VEGF-B increased VEGF signals via VEGF receptor-2 to activate Erk1/2, which resulted in vascular growth. Akt and mTORC1 pathways were upregulated and AMPK downregulated, readjusting cardiomyocyte metabolic pathways to favor glucose oxidation and macromolecular biosynthesis. However, contrasting with a previous theory, there was no difference in fatty acid uptake by the heart between the VEGF-B transgenic, gene-targeted or wildtype rats. Importantly, we also show that VEGF-B expression is reduced in human heart disease. Our data indicate that VEGF-B could be used to increase the coronary vasculature and to reprogram myocardial metabolism to improve cardiac function in ischemic heart disease.

Angiopoietin-2 secretion by endothelial cell exosomes: regulation by the phosphatidylinositol 3-kinase (PI3K)/Akt/endothelial nitric oxide synthase (eNOS) and syndecan-4/syntenin pathways.

Angiopoietin-2 (Ang2) is an extracellular protein and one of the principal ligands of Tie2 receptor that is involved in the regulation of vascular integrity, quiescence, and inflammation. The mode of secretion of Ang2 has never been established, however. Here, we provide evidence that Ang2 is secreted from endothelial cells via exosomes and that this process is inhibited by the PI3K/Akt/endothelial nitric oxide synthase (eNOS) signaling pathway, whereas it is positively regulated by the syndecan-4/syntenin pathway. Vascular defects in Akt1 null mice arise, in part, because of excessive Ang2 secretion and can be rescued by the syndecan-4 knock-out that reduces extracellular Ang2 levels. This novel mechanism connects three critical signaling pathways: angiopoietin/Tie2, PI3K/Akt/eNOS, and syndecan/syntenin, which play important roles in vascular growth and stabilization.