Pericyte-Derived Dickkopf2 Regenerates Damaged Penile Neurovasculature Through an Angiopoietin-1-Tie2 Pathway.

Penile erection requires well-coordinated interactions between vascular and nervous systems. Penile neurovascular dysfunction is a major cause of erectile dysfunction (ED) in patients with diabetes, which causes poor response to oral phosphodiesterase-5 inhibitors. Dickkopf2 (DKK2), a Wnt antagonist, is known to promote angiogenesis. Here, using DKK2-Tg mice or DKK2 protein administration, we demonstrate that the overexpression of DKK2 in diabetic mice enhances penile angiogenesis and neural regeneration and restores erectile function. Transcriptome analysis revealed that angiopoietin-1 and angiopoietin-2 are target genes for DKK2. Using an endothelial cell-pericyte coculture system and ex vivo neurite sprouting assay, we found that DKK2-mediated juxtacrine signaling in pericyte-endothelial cell interactions promotes angiogenesis and neural regeneration through an angiopoietin-1-Tie2 pathway, rescuing erectile function in diabetic mice. The dual angiogenic and neurotrophic effects of DKK2, especially as a therapeutic protein, will open new avenues to treating diabetic ED.

Association of growth factors with arterial recanalization and clinical outcome in patients with ischemic stroke treated with tPA.

SUMMARY BACKGROUND: Growth factors (GF) such as vascular endothelial growth factor (VEGF), angiopoietin-1 (Ang-1) and granulocyte-colony stimulating factor (G-CSF) have been associated with greater efficacy of tissue plasminogen activator (tPA) in experimental studies. OBJECTIVES: To study the association of these GF with arterial recanalization and clinical outcome in patients with acute ischemic stroke treated with tPA. METHODS: We prospectively studied 79 patients with ischemic stroke attributable to MCA occlusion treated with i.v. tPA within the first 3 h from onset of symptoms. Continuous transcranial color-coded sonography (TCCS) was performed during the first 2 h after tPA bolus to assess early MCA recanalization. Hemorrhagic transformation (HT) was classified according to ECASS II definitions. Good functional outcome was defined as a Rankin scale score of 0-2 at 90 days. GF levels were determined by ELISA. RESULTS: Mean serum levels of VEGF, G-CSF and Ang-1 at baseline were significantly higher in patients with early MCA recanalization (n = 30) (all P < 0.0001). In the multivariate analysis, serum levels of VEGF (OR, 1.03), G-CSF (OR, 1.02) and Ang-1 (OR, 1.07) were independently associated with early MCA recanalization (all P < 0.0001). On the other hand, patients with parenchymal hematoma (PH) (n = 20) showed higher levels of Ang-1 (P < 0.0001). Ang-1 (OR, 1.12; P < 0.0001) was independently associated with PH, whereas patients with good outcome (n = 38) had higher levels of G-CSF (P < 0.0001). G-CSF was independently associated with good outcome (OR, 1.12; P = 0.036). CONCLUSIONS: These findings suggest that GF may enhance arterial recanalization in patients with ischemic stroke treated with t-PA, although they might increase the HT.

Oligomerized Tie2 localizes to clathrin-coated pits in response to angiopoietin-1.

The tyrosine kinase receptor Tie2 is expressed on endothelial cells, and together with its ligand angiopoietin-1 (Ang1), is important for angiogenesis and vascular stability. Upon activation by Ang1, Tie2 is rapidly internalized and degraded, a mechanism most likely necessary to attenuate receptor activity. Using immunogold electron microscopy, we show that on the surface of endothelial cells, Tie2 is arranged in variably sized clusters containing dimers and higher order oligomers. Clusters of Tie2 were expressed on the apical and basolateral plasma membranes, and on the tips of microvilli. Upon activation by Ang1, Tie2 co-localized with the clathrin heavy chain at the apical and basolateral plasma membranes and within endothelial cells indicating that Tie2 internalizes through clathrin-coated pits. Inhibiting cellular endocytosis by depleting cellular potassium or by acidifying the cytosol blocked the internalization of Tie2 in response to Ang1. Our results suggest that one pathway mediating the internalization of Tie2 in response to Ang1 is through clathrin-coated pits.

Treatment of stroke with (Z)-1-[N-(2-aminoethyl)-N-(2-ammonioethyl) amino] diazen-1-ium-1, 2-diolate and bone marrow stromal cells upregulates angiopoietin-1/Tie2 and enhances neovascularization.

Neovascularization may contribute to functional recovery after neural injury. Combination treatment of stroke with a nitric oxide donor, (Z)-1-[N-(2-aminoethyl)-N-(2-ammonioethyl) amino] diazen-1-ium-1, 2-diolate (DETA-NONOate) and bone marrow stromal cells promotes functional recovery. However, the mechanisms underlying functional improvement have not been elucidated. In this study, we tested the hypothesis that combination treatment upregulates angiopoietin-1 and its receptor Tie2 in the ischemic brain and bone marrow stromal cells, thereby enhancing cerebral neovascularization after stroke. Adult wild type male C57BL/6 mice were i.v. administered PBS, bone marrow stromal cells 5x10(5), DETA-NONOate 0.4 mg/kg or combination DETA-NONOate with bone marrow stromal cells (n=12/group) after middle cerebral artery occlusion. Combination treatment significantly upregulated angiopoietin-1/Tie2 and tight junction protein (occludin) expression, and increased the number, diameter and perimeter of blood vessels in the ischemic brain compared with vehicle control (mean+ or -S.E., P<0.05). In vitro, DETA-NONOate significantly increased angiopoietin-1/Tie2 protein (n=6/group) and Tie2 mRNA (n=3/group) expression in bone marrow stromal cells. DETA-NONOate also significantly increased angiopoietin-1 protein (n=6/group) and mRNA (n=3/group) expression in mouse brain endothelial cells (P<0.05). Angiopoietin-1 mRNA (n=3/group) was significantly increased in mouse brain endothelial cells treated with DETA-NONOate in combination with bone marrow stromal cell-conditioned medium compared with cells treated with bone marrow stromal cell-conditioned medium or DETA-NONOate alone. Mouse brain endothelial cell capillary tube-like formation assays (n=6/group) showed that angiopoietin-1 peptide, the supernatant of bone marrow stromal cells and DETA-NONOate significantly increased capillary tube formation compared with vehicle control. Combination treatment significantly increased capillary tube formation compared with DETA-NONOate treatment alone. Inhibition of angiopoietin-1 significantly attenuated combination treatment-induced tube formation. Our data indicated that combination treatment of stroke with DETA-NONOate and bone marrow stromal cells promotes neovascularization, which is at least partially mediated by upregulation of the angiopoietin-1/Tie2 axis.