Chemokine localization in bronchial angiogenesis.

Angiogenesis in the lung involves the systemic bronchial vasculature and becomes prominent when chronic inflammation prevails. Mechanisms for neovascularization following pulmonary ischemia include growth factor transit from ischemic parenchyma to upstream bronchial arteries, inflammatory cell migration/recruitment through the perfusing artery, and paracrine effects of lung cells within the left bronchus, the niche where arteriogenesis takes place. We analyzed left lung bronchoalveolar lavage (BAL) fluid and left bronchus homogenates after left pulmonary artery ligation (LPAL) in rats, immediately after the onset of ischemia (0 h), 6 h and 24 h later. Additionally, we tested the effectiveness of dexamethasone on decreasing inflammation (0-24 h LPAL) and angiogenesis at early (3 d LPAL; bronchial endothelial proliferation) and late (14 d LPAL; blood flow) stages. After LPAL (6 h), BAL protein, total inflammatory cells, macrophages, and polymorphonuclear cells increased significantly. In parallel, pro-angiogenic CXC chemokines increased in BAL and the left main-stem bronchus (CXCL1) or only within the bronchus (CXCL2). Dexamethasone treatment reduced total BAL protein, inflammatory cells (total and polymorphonuclear cells), and CXCL1 but not CXCL2 in BAL. By contrast, no decrease was seen in either chemokine within the bronchial tissue, in proliferating bronchial endothelial cells, or in systemic perfusion of the left lung. Our results confirm the presence of CXC chemokines within BAL fluid as well as within the left mainstem bronchus. Despite significant reduction in lung injury and inflammation with dexamethasone treatment, chemokine expression within the bronchial tissue as well as angiogenesis were not affected. Our results suggest that early changes within the bronchial niche contribute to subsequent neovascularization during pulmonary ischemia

Inflammatory cell profile in BAL during the initial 24 h after LPAL.

<p>The total cell number (A) significantly increased at 6 h LPAL (10 rats/group, *P<0.05) then decreased at 24 h LPAL (3 rats/group), returning to baseline (0 h LPAL). The number of macrophages (B) (3–8 rats/group, *P<0.05), as well as the number of neutrophils/PMNs (6–11 rats/group, ***P<0.0001) followed the same trend. Lymphocytes (D) did not change during the same time course (3–7 rats/group). P measured vs 0 h LPAL.</p

Effects of dexamethasone on parenchymal and airway cells and cytokines/receptors.

<p>(A) Summary of the average effect of dexamethasone on measured parameters reflecting changes within the alveolar space. Each bar represents the average % decrease 6 h after LPAL in dexamethasone treated rats compared with vehicle treated rats (3–11 rats/group, *P<0.05). B–C. Average effects of dexamethasone treatment on CXCL1, CXCL2, (mRNA, protein), CXCR1 and CXCR2 (mRNA) expression in the left bronchus 6 h after LPAL. Dexamethasone has no significant effects on any of the variables measured in the bronchus (3–4 rats/group).</p

Time course of total protein in BAL during the initial 24 h following LPAL (BCA assay).

<p>An early increase occurred at 6 h LPAL (20 rats/group, ***P<0.001), and remained elevated at 24 h LPAL (18 rats/group, *P<0.05). Anti-inflammatory treatment with dexamethasone significantly decreased total protein content at 6 h and 24 h LPAL (11–12 rats/time point, ^#P<0.05). *P measured vs 0 h LPAL, ^#P measured vs time-matched untreated.</p

Functional angiogenesis assessed by fluorescent microsphere infusion 14 d after LPAL Dexamethasone treatment had no significant effect on the magnitude of bronchial angiogenesis (n = 4–5/group).

<p>Functional angiogenesis assessed by fluorescent microsphere infusion 14 d after LPAL Dexamethasone treatment had no significant effect on the magnitude of bronchial angiogenesis (n = 4–5/group).</p

CXCR1(A) and CXCR2 (B) mRNA in left and right bronchi, and (C) co-localization of CXCR2 with RECA-1+ subepithelial blood vessel.

<p>Significant changes in CXCR2 were measured only in the left bronchus (*P<0.05 from 0 h and ##P<0.01 from right bronchus). Frozen sections of left bronchus 6 h after LPAL show co-localization of anti-CXCR2 (red) with RECA-1+ subepithelial blood vessels (green: 100× original magnification, and inset 600× original magnification).</p

CXCL1 and CXCL2 cytokines mRNA (A, B), protein levels in the left bronchus (C), and (D) frozen section of left bronchus 6 h after LPAL with double staining for CXCL2 (red) and the epithelial cell marker Epcam (green;100× original magnification and inset: 600× original magnification).

<p>CXCL1 (A) and CXCL2 (B) mRNA and CXCL1 and CXCL2 protein levels (C) increased at 6 h LPAL (*P<0.05) and returned to baseline by 24 h LPAL (3–4 rats/time point). Co-localization of stain for epithelial cells (Epcam,;green) and anti-CXCL2 (red) suggest the airway epithelium is a prominent source for CXCL2.</p

Changes in proliferating bronchial vessels.

<p>(A) Histologic section of airway demonstrating bronchial vessels by H&E (left panel) and serial section stained for PCNA (right panel). Bronchial vessels show abundant PCNA+ endothelial cells. (B) After LPAL (3 d), a significant increase in the fraction of PCNA⁺ bronchial vessels is observed. Treatment with dexamethasone had no significant effect on this proliferation index. Control includes both sham and right lungs (3–5 rats/group, **P<0.01 vs control).</p

Ascorbic acid promotes cardiomyogenesis through SMAD1 signaling in differentiating mouse embryonic stem cells

<div><p>Numerous groups have documented that Ascorbic Acid (AA) promotes cardiomyocyte differentiation from both mouse and human ESCs and iPSCs. AA is now considered indispensable for the routine production of hPSC-cardiomyocytes (CMs) using defined media; however, the mechanisms involved with the inductive process are poorly understood. Using a genetically modified mouse embryonic stem cell (mESC) line containing a dsRED transgene driven by the cardiac-restricted portion of the <i>ncx1</i> promoter, we show that AA promoted differentiation of mESCs to CMs in a dose- and time-dependent manner. Treatment of mPSCs with AA did not modulate total SMAD content; however, the phosphorylated/active forms of SMAD2 and SMAD1/5/8 were significantly elevated. Co-administration of the SMAD2/3 activator Activin A with AA had no significant effect, but the addition of the nodal co-receptor TDGF1 (Cripto) antagonized AA’s cardiomyogenic-promoting ability. AA could also reverse some of the inhibitory effects on cardiomyogenesis of ALK/SMAD2 inhibition by SB431542, a TGFβ pathway inhibitor. Treatment with BMP2 and AA strongly amplified the positive cardiomyogenic effects of SMAD1/5/8 in a dose-dependent manner. AA could not, however, rescue dorsomorphin-mediated inhibition of ALK/SMAD1 activity. Using an inducible model system, we found that SMAD1, but not SMAD2, was essential for AA to promote the formation of TNNT2⁺-CMs. These data firmly demonstrate that BMP receptor-activated SMADs, preferential to TGFβ receptor-activated SMADs, are necessary to promote AA stimulated cardiomyogenesis. AA-enhanced cardiomyogenesis thus relies on the ability of AA to modulate the ratio of SMAD signaling among the TGFβ-superfamily receptor signaling pathways.</p></div

Ascorbic acid-mediated cardiogenic-induction is independent of its anti-oxidant and anti-proliferative capacities.

<p><b>A.</b> Cardiomyogenesis (RFP fluorescence) assessed in RFP6-EBs at Day 7+3 after treatment with AA (100 μM), Vitamin E (100 μM) and NAC (1000 μM) (n = 3) performed at Day 2 of differentiation. <b>B.</b> Analysis of DNA content (PI staining) of dissociated EBs at Day 3 of differentiation, 24 hours after treatment with AA (Day 2, 1000 μM, n = 3). The histograms show cell cycle stages G0/G1, S and G2/M. No difference in the percentage of cells could be demonstrated between the treated and untreated controls. *p<0.05 compared to untreated control.</p