Critical Role of Endothelial Hydrogen Peroxide in Post-Ischemic Neovascularization

<div><p>Background</p><p>Reactive oxygen species (ROS) play an important role in angiogenesis in endothelial cells (ECs) <i>in vitro</i> and neovascularization <i>in vivo</i>. However, little is known about the role of endogenous vascular hydrogen peroxide (H₂O₂) in postnatal neovascularization.</p> <p>Methodology/Principal Findings</p><p>We used Tie2-driven endothelial specific catalase transgenic mice (Cat-Tg mice) and hindlimb ischemia model to address the role of endogenous H₂O₂ in ECs in post-ischemic neovascularization <i>in vivo</i>. Here we show that Cat-Tg mice exhibit significant reduction in intracellular H₂O₂ in ECs, blood flow recovery, capillary formation, collateral remodeling with larger extent of tissue damage after hindlimb ischemia, as compared to wild-type (WT) littermates. In the early stage of ischemia-induced angiogenesis, Cat-Tg mice show a morphologically disorganized microvasculature. Vascular sprouting and tube elongation are significantly impaired in isolated aorta from Cat-Tg mice. Furthermore, Cat-Tg mice show a decrease in myeloid cell recruitment after hindlimb ischemia. Mechanistically, Cat-Tg mice show significant decrease in eNOS phosphorylation at Ser1177 as well as expression of redox-sensitive vascular cell adhesion molecule-1 (VCAM-1) and monocyte chemotactic protein-1 (MCP-1) in ischemic muscles, which is required for inflammatory cell recruitment to the ischemic tissues. We also observed impaired endothelium-dependent relaxation in resistant vessels from Cat-Tg mice.</p> <p>Conclusions/Significance</p><p>Endogenous ECs-derived H₂O₂ plays a critical role in reparative neovascularization in response to ischemia by upregulating adhesion molecules and activating eNOS in ECs. Redox-regulation in ECs is a potential therapeutic strategy for angiogenesis-dependent cardiovascular diseases.</p> </div

Endothelial catalase overexpression blunts endothelium-dependent relaxation of resistant vessels.

<p><b>A</b>, the first branches mesenteric arteries were harvested from Wild-type (WT) and Tie2-driven catalase transgenic (Cat-Tg) mice and assessed for endothelium-dependent or – independent relaxation by acetylcholine or sodium nitroprusside, respectively (n = 4 per group and *p<0.05). Data shown are mean+SE. <b>B</b>, a proposed model for the role of endogenous H₂O₂ in endothelial cells during ischemia-induced neovascularization. Tissue ischemia induces endogenous reactive oxygen species production including hydrogen peroxide (H₂O₂) intracellularly and extracellularly for endothelial cells. Intracellular H₂O₂, which can be reduced by Tie2-driven catalase overexpression in this study, promote crucial neovascular signaling regulating endothelial sprouting and tube formation, endothelial nitric oxide synthase (eNOS) activation as well as the expression of vascular adhesion molecule (VCAM)-1 and monocyte chemoattractant protein (MCP)-1. Endothelial H₂O₂ could be involved in vascular progenitor mobilization. H₂O₂ is thought to be diffusible across cellular membrane (blue arrow). Myeloid recruitment, vascular endothelial growth factor (VEGF) and potential nitric oxide (NO) production are regulated by endogenous H₂O₂ in endothelial cells during neovascularization.</p

Endothelial catalase overexpression impairs vessel sprouting and tube elongation in <i>ex vivo</i> aortic ring assay.

<p>Aortas were harvested from Wild-type (WT) and Tie2-driven catalase transgenic (Cat-Tg) mice and cultured in Matrigel for 7 days. Capillary sprouts and average tube length were measured in 5 rings from each aorta under microscopy (n = 3 mice per group and *p<0.05). Data shown are mean+SE.</p

Intracellular H₂O₂ in endothelial cells regulate endothelial nitric oxide synthase activation <i>in vivo</i>.

<p><b>A</b>, intracellular redox status was measured by 2′,7′-dichlorfluorescein-diacetate (DCF-DA) staining in gated CD31⁺/CD45⁻ population of collagenase-digested ischemic muscles at day 3. The dotted lines indicate the background signals without DCF-DA. <b>B</b>, ischemic muscles from Wild-type (WT) and Tie2-driven catalase transgenic (Cat-Tg) mice at day 3 were isolated and incubated. Their H₂O₂ production was measured by Amplex Ultra Red assay. <b>C</b>, harvested ischemic and non-ischemic muscles at day 3 were analyzed for protein expression of phosphorylated and total form of endothelial nitric oxide synthase (eNOS), Akt and ERK1/2 (as control) by Western analysis. Densitometry analysis in activation (phosphorylation) of each protein is shown. All data shown are mean+SE (n = 3 mice per group, *p<0.05, **p<0.01 and ***p<0.001).</p

Endothelial catalase overexpression decreases the recruitment of F4/80+ myeloid cells to the ischemic tissue.

<p><b>A</b>, the ischemic area of gastrocnemius muscles from wild-type (WT) and Tie2-driven catalase transgenic (Cat-Tg) mice at day 7 was analyzed for myeloid cell recruitment with immunostaining for F4/80 (brown and arrows). The percentage of F4/80+ cell infiltrated area in the damaged region of gastrocnemius muscles is shown (n = 3 mice per group). <b>B</b>, adductor muscles in the upper limb were harvested at day 3 and analyzed for F4/80+ myeloid accumulation (brown and arrows) at the perivascular space of collateral arteries. Eosin staining was performed to show the structures. (n = 3 mice per group and *p<0.05). <b>C</b>, ischemic tibialis anterior muscles were harvested at day 3 and analyzed for mRNA expression of intercellular adhesion molecule 1 (<i>icam1</i>), vascular cell adhesion molecule 1 (<i>vcam1</i>) and monocyte chemotactic protein-1 (MCP-1 (<i>ccl2</i>)) by real-time polymerase chain reaction. Ribosomal 18S and <i>hprt</i> were used as internal controls. Relative expression for WT is shown (n = 3 mice per group). <b>D</b>, vascular endothelial growth factor (VEGF) expression was analyzed by Western blotting of protein lysate from ischemic tibialis anterior muscle at day 7. Alpha tubulin is shown as control. Densitometry analysis is shown (n = 3 mice per group). All data shown are mean+SE (*p<0.05).</p

Endothelial catalase overexpression impairs collateral remodeling and stabilization of vessels undergoing neovascularization.

<p><b>A</b>, collateral remodelling after hindlimb ischemia was analyzed in the same anatomical arteries localized at semimembranosus muscles in the upper limbs between wild-type (WT) and Tie2-driven catalase transgenic (Cat-Tg) mice. The luminal diameter and wall area are calculated from the measurements of luminal and perivascular tracing. Arrows indicate collateral wall. <b>B</b>, ischemic gastrocnemius muscles were analyzed for the morphology of vessels at day 3 with immunostaining for an endothelial-marker, CD31 (green). Nuclei were visualized by 4′,6-diamidino-2-phenylindole (DAPI) (blue). Magnified images show that morphologically disorganized vessels with varying size, enlarged lumen and irregular shape are often seen in Cat-Tg mice. C, capillary densities in the same region as B were analyzed by CD31 staining at day 7. Bars indicate 20 μm in A, and 50 μm in B and C. All data shown are mean+SE (n = 3–4 mice per group, *p<0.05 and **p<0.01).</p

Tie2-driven catalase overexpression affects circulating progenitor cell level.

<p>The levels of white blood cells, monocytes and vascular progenitors (Sca1⁺/Flk1⁺ cells) were analyzed in the peripheral blood at indicated time points. Representative plots of vascular progenitors at day 2 are shown. All data shown are mean+SE (n = 4 mice per group and *p<0.05).</p

Flk1+ and VE-Cadherin+ Endothelial Cells Derived from iPSCs Recapitulates Vascular Development during Differentiation and Display Similar Angiogenic Potential as ESC-Derived Cells

<div><p>Rationale</p><p>Induced pluripotent stem (iPS) cells have emerged as a source of potentially unlimited supply of autologous endothelial cells (ECs) for vascularization. However, the regenerative function of these cells relative to adult ECs and ECs derived from embryonic stem (ES) cells is unknown. The objective was to define the differentiation characteristics and vascularization potential of Fetal liver kinase (Flk)1⁺ and Vascular Endothelial (VE)-cadherin⁺ ECs derived identically from mouse (m)ES and miPS cells. </p> <p>Methods and Results</p><p>Naive mES and miPS cells cultured in type IV collagen (IV Col) in defined media for 5 days induced the formation of adherent cell populations, which demonstrated similar expression of Flk1 and VE-cadherin and the emergence of EC progenies. FACS purification resulted in 100% Flk1⁺ VE-cadherin⁺ cells from both mES and miPS cells. Emergence of Flk1⁺VE-cadherin⁺ cells entailed expression of the vascular developmental transcription factor <i>Er71</i>, which bound identically to <i>Flk1, VE-cadherin</i>, and <i>CD31</i> promoters in both populations. Immunostaining with anti-VE-cadherin and anti-CD31 antibodies and microscopy demonstrated the endothelial nature of these cells. Each cell population (unlike mature ECs) organized into well-developed vascular structures <i>in</i><i>vitro</i> and incorporated into CD31⁺ neovessels in matrigel plugs implanted in nude mice <i>in</i><i>vivo</i>.</p> <p>Conclusion</p><p>Thus, iPS cell-derived Flk1⁺VE-cadherin⁺ cells expressing the Er71 are as angiogenic as mES cell-derived cells and incorporate into CD31⁺ neovessels. Their vessel forming capacity highlights the potential of autologous iPS cells-derived EC progeny for therapeutic angiogenesis.</p> </div

Angiogenic potential of Flk1⁺VE-cadherin⁺ ECs derived from mES and iPS cells.

<p>Quantification of branching point structures; control mouse ECs and mES- or iPS-derived Flk1⁺VE-cadherin⁺ ECs (2x10⁵) were plated onto Matrigel in the presence of VEGF¹⁶⁵ (50 ng/ml). After 18 hr, the numbers of branching points were counted. Data are expressed as percentage of branching points (n=3, *P <0.05 vs. control or as indicated) (A); Representative phase contrast images of branching point structures. The experiments were repeated 3 times with the use of triplicate wells. Scale bar, 200 µm. The arrows indicate branching points (B-G). </p

Mouse iPS-derived Flk1⁺VE-cadherin⁺ cells incorporate into CD31-positive vessels.

<p>Time line of the Matrigel experiment (A). Representative images of H&E stained Matrigel plugs obtained from nude mice receiving control mouse ECs or mES- or iPS-derived Flk1⁺VE-cadherin⁺ ECs (2x10⁵) (see Methods for details) (B,C,D). Immunohistochemistry of indicating that Matrigel sections were stained with anti-CD31 (green) and mCherry (red, arrowheads) (E-M). Autofluorescent leukocytes indicated perfused vessels (L). Quantification of number of neovessels in Matrigel sections incorporating the Flk1⁺VE-cadherin⁺ cells expressing mCherry (N). Scale bar 100 µm. Experiments were repeated at least 3 times. </p