Endothelial E-selectin potentiates neovascularization via endothelial progenitor cell-dependent and -independent mechanisms.

BACKGROUND: Although potential participation of bone marrow-derived circulating endothelial progenitor cells (EPCs) to neoangiogenesis has been proposed, the precise molecular mechanisms of EPC recruitment to vascular endothelium has not been fully elucidated. METHODS AND RESULTS: Peripheral blood mononuclear cells were isolated from healthy volunteers and cultured for 7 days to obtain EPCs. Tumor necrosis factor-alpha-activated human umbilical vein endothelial cells (HUVECs) supported significantly more rolling and adhesion of EPCs compared with inactivated HUVEC monolayer. Pretreatment of activated HUVEC with an adhesion-blocking mAb to E-selectin significantly reduced EPCs adhesion to HUVECs. When HUVECs were transduced with a recombinant adenovirus of E-selectin (AdRSVE-sel) or that of beta-galactosidase (AdRSVLacZ), E-selectin-transduced but not LacZ-transduced HUVECs exhibited significantly more EPC rolling as well as adhesion. Further, effect of AdRSVE-sel or AdRSVLacZ was examined in mouse hind limb ischemic model. AdRSVE-sel-transduced mice showed significantly less limb necrosis and higher laser Doppler ratio when compared with AdRSVLacZ-transduced mice. Interestingly, blood flow recovery of ischemic limb observed in AdRSVE-sel-transduced mice was more prominent when combined with EPC administration compared with that of AdRSVLacZ-transduced mice. CONCLUSIONS: Endothelial E-selectin plays a crucial role in EPC-endothelial interaction in vitro. The importance of E-selectin was also confirmed in vivo even in the absence of exogenous EPC. These data provide molecular background for novel cell-based therapy for ischemic atherosclerosis.

Targeted delivery of bone marrow mononuclear cells by ultrasound destruction of microbubbles induces both angiogenesis and arteriogenesis response.

OBJECTIVE: Ultrasound (US)-mediated destruction of contrast microbubbles causes capillary rupturing that stimulates arteriogenesis, whereas intramuscular implantation (im) of bone marrow mononuclear cells (BM-MNCs) induces angiogenesis. We therefore studied whether US-targeted microbubble destruction combined with transplantation of BM-MNCs can enhance blood flow restoration by stimulating both angiogenesis and arteriogenesis. METHODS AND RESULTS: US-mediated destruction of phospholipid-coated microbubbles was applied onto ischemic hindlimb muscle and subsequently BM-MNCs were transfused. A significant enhancement in blood flow recovery after Bubble+US+BM-MNC infusion (34% increase, P<0.05) was observed compared with Bubble+US (25%). The ratio of capillary/muscle fiber increased by Bubble+US+BM-MNC-i.v (260%, P<0.01) than that in the Bubble+US group (172%), into which BM-MNCs were incorporated (angiogenesis). Smooth muscle alpha-actin-positive arterioles were also increased, and angiography showed augmented collateral vessel formation (arteriogenesis). Platelet-derived proinflammatory factors activated by Bubble+US induces the expression of adhesion molecules (P-selectin and ICAM-1), leading to the attachment of transplanted BM-MNCs on the endothelium. Flow assay confirmed that the platelet-derived factors cause the adhesion of BM-MNCs onto endothelium under laminar flow. CONCLUSIONS: This study demonstrates that the targeted delivery of BM-MNCs by US destruction of microbubbles enhances regional angiogenesis and arteriogenesis response, in which the release of platelet-derived proinflammatory factors activated by Bubble+US play a key role in the attachment of transplanted BM-MNCs onto the endothelial layer.

Blockade of cell adhesion by a small molecule selectin antagonist attenuates myocardial ischemia/reperfusion injury.

Reperfusion injury is related closely to inflammatory reactions such as the activation and accumulation of neutrophils. We investigated the efficacy of a novel small molecule selectin antagonist (bimosiamose) in a rat model of transient left coronary artery occlusion (30 min) and reperfusion (24 h). Treatment with bimosiamose (25 mg/kg, intravenously at reperfusion) showed a significant reduction in infarction area/area at risk of approximately 41% compared to vehicle control (P=0.01) and preserved the left ventricular function. The accumulation of polymorphonuclear neutrophils at the site of area at risk was decreased significantly, accompanied by 78% reduction of the myeloperoxidase activity. Parallel-plate flow chamber analysis revealed that bimosiamose showed a significant inhibition in rolling (62%, P<0.001) and adhesion (38%, P<0.05) of HL-60 cells to activated human umbilical vein endothelial cells compared with vehicle control. This study demonstrates for the first time that bimosiamose, a novel small molecule selectin antagonist, attenuates significantly ischemia/reperfusion injury.

Introduction of short interfering RNA to silence endogenous E-selectin in vascular endothelium leads to successful inhibition of leukocyte adhesion.

Short interfering RNAs (siRNAs) are powerful sequence-specific reagents that suppress gene expression in mammalian cells. We report for the first time that gene silencing of endothelial E-selectin by siRNAs leads to successful inhibition of leukocyte-endothelial interaction under flow. siRNAs designed to target human E-selectin were tranfected into human umbilical vein endothelial cells (HUVEC). Western blotting analysis revealed that transfection of these siRNAs, but not the scrambled control siRNA (100nM each), attenuated E-selectin expression in HUVEC activated with TNF-alpha (10ng/ml, 4h) without affecting expression of ICAM-1. Moreover, a leukocyte adhesion assay under flow (shear stress=1.0dyne/cm(2)) demonstrated that HUVEC transfected with a siRNA against E-selectin (siE-01) supported significantly less HL60 adhesion as compared to those transfected with the control siRNA (scE-01) after activation (p<0.03). This technique provides a powerful strategy to dissect a specific function of a given molecule in leukocyte-endothelial interaction.

Bone marrow monocyte lineage cells adhere on injured endothelium in a monocyte chemoattractant protein-1-dependent manner and accelerate reendothelialization as endothelial progenitor cells.

Peripheral blood (PB)-derived CD14+ monocytes were shown to transdifferentiate into endothelial cell (EC) lineage cells and contribute to neovascularization. We investigated whether bone marrow (BM)- or PB-derived CD34-/CD14+ cells are involved in reendothelialization after carotid balloon injury. Although neither hematopoietic nor mesenchymal stem cells were included in human BM-derived CD34-/CD14+ monocyte lineage cells (BM-MLCs), they expressed EC-specific markers (Tie2, CD31, VE-cadherin, and endoglin) to an extent identical to mature ECs. When BM-MLCs were cultured with vascular endothelial growth factors, hematopoietic markers were drastically decreased and new EC-specific markers (Flk and CD34) were induced. BM-MLCs were intra-arterially transplanted into balloon-injured arteries of athymic nude rats. When BM-MLCs were activated by monocyte chemoattractant protein-1 (MCP-1) in vivo or in vitro, they adhered onto injured endothelium, differentiated into EC-like cells by losing hematopoietic markers, and inhibited neointimal hyperplasia. Ability to prevent neointimal hyperplasia was more efficient than that of BM-derived CD34+ cells. MCP-dependent adhesion was not observed in PB-derived CD34-/CD14+ monocytes. Regenerated endothelium exhibited a cobblestone appearance, blocked extravasation of dye, and induced NO-dependent vasorelaxation. Basal adhesive activities on HUVECs under laminar flow and beta1-integrin expression (basal and active forms) were significantly increased in BM-MLCs compared with PB-derived monocytes. MCP-1 markedly enhanced adhesive activity of BM-MLCs (2.8-fold) on HUVECs by activating beta1-integrin conformation. Thus, BM-MLCs can function as EC progenitors that are more potent than CD34+ cells and acquire the ability to adhere on injured endothelium in a MCP-1-dependent manner, leading to reendothelialization associated with inhibition of intimal hyperplasia. This will open a novel window to MCP-1-mediated biological actions and vascular regeneration strategies by cell therapy.