Enhanced hemangioblast generation and improved vascular repair and regeneration from embryonic stem cells by defined transcription factors

SummaryThe fetal liver kinase 1 (FLK-1)+ hemangioblast can generate hematopoietic, endothelial, and smooth muscle cells (SMCs). ER71/ETV2, GATA2, and SCL form a core transcriptional network in hemangioblast development. Transient coexpression of these three factors during mesoderm formation stage in mouse embryonic stem cells (ESCs) robustly enhanced hemangioblast generation by activating bone morphogenetic protein (BMP) and FLK-1 signaling while inhibiting phosphatidylinositol 3-kinase, WNT signaling, and cardiac output. Moreover, etsrp, gata2, and scl inhibition converted hematopoietic field of the zebrafish anterior lateral plate mesoderm to cardiac. FLK-1+ hemangioblasts generated by transient coexpression of the three factors (ER71-GATA2-SCL [EGS]-induced FLK-1+) effectively produced hematopoietic, endothelial, and SMCs in culture and in vivo. Importantly, EGS-induced FLK-1+ hemangioblasts, when codelivered with mesenchymal stem cells as spheroids, were protected from apoptosis and generated functional endothelial cells and SMCs in ischemic mouse hindlimbs, resulting in improved blood perfusion and limb salvage. ESC-derived, EGS-induced FLK-1+ hemangioblasts could provide an attractive cell source for future hematopoietic and vascular repair and regeneration

The peroxisome proliferator-activated receptor agonist pioglitazone and 5-lipoxygenase inhibitor zileuton have no effect on lung inflammation in healthy volunteers by positron emission tomography in a single-blind placebo-controlled cohort study.

BACKGROUND:Anti-inflammatory drug development efforts for lung disease have been hampered in part by the lack of noninvasive inflammation biomarkers and the limited ability of animal models to predict efficacy in humans. We used 18F-fluorodeoxyglucose (18F-FDG) positron emission tomography (PET) in a human model of lung inflammation to assess whether pioglitazone, a peroxisome proliferator-activated receptor-γ (PPAR-γ) agonist, and zileuton, a 5-lipoxygenase inhibitor, reduce lung inflammation. METHODS:For this single center, single-blind, placebo-controlled cohort study, we enrolled healthy volunteers sequentially into the following treatment cohorts (N = 6 per cohort): pioglitazone plus placebo, zileuton plus placebo, or dual placebo prior to bronchoscopic endotoxin instillation. 18F-FDG uptake pre- and post-endotoxin was quantified as the Patlak graphical analysis-determined Ki (primary outcome measure). Secondary outcome measures included the mean standard uptake value (SUVmean), post-endotoxin bronchoalveolar lavage (BAL) cell counts and differentials and blood adiponectin and urinary leukotriene E4 (LTE4) levels, determined by enzyme-linked immunosorbent assay, to verify treatment compliance. One- or two-way analysis of variance assessed for differences among cohorts in the outcome measures (expressed as mean ± standard deviation). RESULTS:Ten females and eight males (29±6 years of age) completed all study procedures except for one volunteer who did not complete the post-endotoxin BAL. Ki and SUVmean increased in all cohorts after endotoxin instillation (Ki increased by 0.0021±0.0019, 0.0023±0.0017, and 0.0024±0.0020 and SUVmean by 0.47±0.14, 0.55±0.15, and 0.54±0.38 in placebo, pioglitazone, and zileuton cohorts, respectively, p<0.001) with no differences among treatment cohorts (p = 0.933). Adiponectin levels increased as expected with pioglitazone treatment but not urinary LTE4 levels as expected with zileuton treatment. BAL cell counts (p = 0.442) and neutrophil percentage (p = 0.773) were similar among the treatment cohorts. CONCLUSIONS:Endotoxin-induced lung inflammation in humans is not responsive to pioglitazone or zileuton, highlighting the challenge in translating anti-inflammatory drug efficacy results from murine models to humans. TRIAL REGISTRATION:ClinicalTrials.gov NCT01174056

Mean standard uptake value (SUV) results from¹⁸F-fluorodeoxyglucose PET images in the right and left lungs for each treatment cohort.

<p>Arrows indicate presence of both Asp299Gly and Thr399Ile single nucleotide polymorphisms (SNPs), arrowheads only the Asp299Gly SNP. The arrow and arrowhead for the left lung of the placebo cohort point to the top two post-endotoxin data points. * = p < 0.05 when comparing post-endotoxin (After) to pre-endotoxin (Before) value.</p

Baseline clinical characteristics.

<p>Baseline clinical characteristics.</p

Positron emission tomography (PET) and computed tomography (CT) images from a representative volunteer in each treatment cohort.

<p>White outlines show the volumes of interest (VOIs) used to determine the time-activity curves for the Patlak graphical analysis and standard uptake values. VOIs were sometimes smaller in volume on the left due to the heart.</p

Study design, participant and procedure flow.

<p>Reasons for screen failures were: Elevated alanine aminotransferase level (N = 5); % predicted forced expiratory volume in one second (FEV1) and/or forced vital capacity (FVC) < 90% (N = 6); minimal airway obstruction on pulmonary function testing (N = 1); abnormal chest x-ray (N = 1); abnormal electrocardiogram (N = 1); history of childhood asthma (N = 1); smoking within 1 year of enrollment (N = 1); body mass index outside range (N = 1). Withdrawals were volunteers who signed the consent after completing the screening procedures and were given the blinded study drugs but withdrew from the study prior to initiating the imaging and bronchoscopy procedures and were lost to follow up. FDG-PET = positron emission tomography imaging with ¹⁸F-fluorodeoxyglucose. i.b. = intrabronchial. qD = once per day. Q6hr = once every six hours.</p

BAL cell counts and differentials.

<p>BAL cell counts and differentials.</p

Patlak graphical analysis results from¹⁸F-fluorodeoxyglucose (¹⁸F-FDG) PET images in the right and left lungs before and after endotoxin instillation.

<p>Arrows indicate presence of both Asp299Gly and Thr399Ile single nucleotide polymorphisms (SNPs), arrowheads only the Asp299Gly SNP. The arrowhead for the left lung data of the zileuton treatment cohort points to the post-endotoxin data point that decreased slightly after endotoxin. <i>K</i>_i = influx constant describing rate of ¹⁸F-FDG uptake into the lung region of interest, determined by Patlak graphical analysis. * = p < 0.05 when comparing post-endotoxin (After) to pre-endotoxin (Before) value.</p

Effect of endotoxin on clinical characteristics.

<p>Effect of endotoxin on clinical characteristics.</p