Runxia Tian

Stable Compounds of Cigarette Smoke Induce Endothelial Superoxide Anion Production via NADPH Oxidase Activation

Objective—Endothelial dysfunction is an early manifestation of cigarette smoke (CS) toxicity. We have previously demonstrated that CS impairs nitric oxide (NO)-mediated endothelial function via increased generation of superoxide anion (SYMBOL). In these studies, we investigated whether stable compounds present in CS activate specific pathways responsible for the increased endothelial SYMBOL production. Methods and Results—Short exposure of bovine pulmonary artery endothelial cells (BPAECs), human pulmonary artery endothelial cells, and rat pulmonary arteries to CS extracts (CSEs) resulted in a large increase in SYMBOL production (20-fold, 3-fold, and 2-fold increase, respectively; P< 0.05 versus control), which was inhibited by the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase inhibitors diphenyleneiodinium, apocynin, and gp91 docking sequencetat peptide but not by oxypurinol, the NO synthase inhibitor NG-nitro-L-arginine methyl ester, or the mitochondrial respiration inhibitor rotenone. Exposure of BPAECs to acrolein, a stable thiol-reactive agent found in CS, increased SYMBOL production 5-fold, which was prevented by prior inhibition of NADPH oxidase. Conclusions—These studies demonstrate that thiol-reactive stable compounds in CS can activate NADPH oxidase and increase endothelial SYMBOL production, thereby reducing NO bioactivity and resulting in endothelial dysfunction. Clinically, these studies may contribute to the development of agents able to mitigate CS-mediated vascular toxicity.

Notch activation induces endothelial cell senescence and pro-inflammatory response: implication of Notch signaling in atherosclerosis.

OBJECTIVE Notch signaling plays pivotal roles in the pathogenesis of vascular disease. However, little is known about its role in atherosclerosis. We sought to investigate the potential involvement of the Notch signaling in atherosclerosis. METHODS Expression of Notch pathway components in mouse and human aorta with or without atherosclerosis plaque was examined by immunohistochemistry. Expression of Notch target genes in young versus aged human endothelial cells (EC) was examined by PCRArray and immunoblot. In vitro loss- and gain-of-function approaches were utilized to evaluate the role of Notch signaling in inducing EC senescence and secretion of pro-inflammatory cytokines by ProteinArray. Notch gene profile was studied in 1054 blood samples of patients with coronary artery disease (CAD). Genotyping was performed using the Genome-Wide Single Nucleotide Polymorphism (SNP) Array. RESULTS Notch pathway components were upregulated in luminal EC at atherosclerotic lesions from mouse and human aortas. In addition, the Notch pathway was activated in aged but not young human EC. Enforced Notch activation resulted in EC senescence and significantly upregulated expression of several molecules implicated in the inflammatory response (IL-6/IL-8/IL-1α/RANTES/ICAM-1). The upregulated IL-6 was partially responsible for mediating leukocyte transendothelial migration. Genetic association analysis detected, of 82 SNPs across 6 Notch pathway genes analyzed, 4 SNPs with nominal association with CAD burden. CONCLUSION Notch pathway is activated in luminal EC at atherosclerotic plaques and results in pro-inflammatory response and senescence of EC. Notch signaling may be linked to human CAD risk. These findings implicate a potential involvement of Notch signaling in atherosclerosis.

A Novel Autologous Cell Based Therapy to Promote Diabetic Wound Healing

Objectives:We have previously shown that stromal cell–derived factor-1&agr; (SDF-1&agr;) is downregulated within diabetic cutaneous wounds, and that direct application of recombinant SDF-1&agr; increases wound closure rates, neovascularization, and endothelial progenitor cell (EPC) recruitment. However, increased wound levels of exogenous SDF-1&agr; results in elevated systemic levels of this proangiogenic chemokine that raises concerns for tumorigenesis and inflammation. We now seek to test the efficacy of a novel, safer cell-based therapy (CBT) employing ex vivo primed bone marrow-derived stem cells (BMDSC) with SDF-1&agr;. We also elucidate the mechanism of action of this new approach for accelerating diabetic wound healing. Methods:Unfractionated BMDSC from diabetic Leprdb/db mice were incubated for 20 hours with SDF-1&agr; (100 ng/mL) or bovine serum albumin (control). Pretreated BMDSC (1 × 106) were injected subcutaneously into full-thickness skin wounds in Leprdb/db mice (n = 8 per group). Wound closure rates, capillary density, and the recruitment of EPC were assessed with serial photography, DiI perfusion, confocal microscopy, and immunohistochemistry. The expression of molecular targets, which may mediate prohealing/proangiogenic effects of SDF-1&agr;–primed BMDSC was evaluated by polymerase chain reaction array and immunoblotting assay. The biological function of a potential mediator was tested in a mouse wound-healing model. Serum SDF-1&agr; levels were measured with enzyme-linked immunosorbent assay (ELISA). Results:SDF-1&agr;–primed BMDSC significantly promote wound healing (P < 0.0001), neovascularization (P = 0.0028), and EPC recruitment (P = 0.0059). Gene/protein expression studies demonstrate upregulation of Ephrin Receptor B4 and plasminogen as downstream targets potentially mediating the prohealing and proangiogenic responses. Ex vivo BMDSC activation and the subsequent inoculation of cells into wounds does not increase systemic SDF-1&agr; levels. Conclusions:We report a novel CBT that is highly effective in promoting healing and neovascularization in a murine model of type 2 diabetes. Furthermore, we identify new molecular targets that may be important for advancing the field of wound healing.

Identification of E-selectin as a novel target for the regulation of postnatal neovascularization: Implications for diabetic wound healing

Objectives:We previously reported that stromal cell-derived factor-1&agr; (SDF-1&agr;, a homing signal for recruiting endothelial progenitor cells (EPC) to areas of neovascularization), is down-regulated in diabetic wounds (Gallagher et al, J Clin Invest. 2007;117:1249–1259). We now investigate signals whereby mature endothelial cells (EC) and circulating EPC achieve SDF-1&agr;-mediated EPC homing. Methods:SDF-1&agr; in diabetic wounds were therapeutically increased by injection of SDF-1&agr;-engineered bone marrow-derived fibroblasts versus control cells (N = 48 [20, non-obese diabetic (NOD)], [28, streptozotocin-C57]). Polymerase chain reaction-array gene expression differences were validated by Western blotting and immunohistochemistry. The role of adhesion molecule(s) in mediating SDF-1&agr;-induced EPC homing, and wound healing was furthered studied using antagonists in vitro and in vivo. Results:Increasing wound SDF-1&agr; via cell-based therapy promotes healing in diabetic mice (∼20% increase in healing rates by day 3, P = 0.006). SDF-1&agr; increased EC-EPC adhesion and specifically upregulated E-selectin expression in human microvascular EC (2.3-fold increase, P < 0.01). This effect was also significant in blood vessels of the experimental mice and resulted in increased wound neovascularization. The regulatory effects of SDF-1&agr; on EC-EPC adhesion and EPC homing were specifically mediated by E-selectin, as the application of E-selectin antagonists significantly inhibited SDF-1&agr;-induced EC-EPC adhesion, EPC homing, wound neovascularization, and wound healing. Conclusions:SDF-1&agr;-engineered cell-based therapy promotes diabetic wound healing in mice by specifically upregulating E-selectin expression in mature EC leading to increase EC-EPC adhesion, EPC homing, and increased wound neovascularization. These findings provide novel insight into the signals underlying the biological effect of SDF-1&agr; on EPC homing and point to E-selectin as a new potential target for therapeutic manipulation of EPC trafficking in diabetic wound healing.

Inhibition of tumor angiogenesis and melanoma growth by targeting vascular E-selectin.

Objectives:Aggressive human melanomas express, C-X-C chemokine receptor 4 (CXCR4), the receptor for the chemokine, stromal cell-derived factor-1alpha (SDF-1&agr;). The CXCR4-SDF-1&agr; axis has been postulated to increase melanoma invasiveness. We discovered that SDF-1&agr; specifically upregulates E-selectin on endothelial cells, thus tethering circulating endothelial progenitor cells (EPC) and facilitating homing. We investigated the hypothesis that small interfering ribonucleic acid (siRNA)-mediated E-selectin blockade inhibits melanoma angiogenesis and tumor growth. Methods:Human melanoma cells overexpressing SDF-1&agr; were xenografted on severe combined immunodeficiency (SCID) mice. SDF-1&agr; expression in cells was measured by enzyme-linked immunosorbent assay (ELISA). In vitro melanoma cell growth was examined by cell proliferation assay. In vivo vascular E-selectin knockdown was achieved by administration of high-volume E-selectin siRNA (100 pmol/180 &mgr;L/week × 3 times) and inhibition was validated by immunostaining (N = 6/group, E-Selectin siRNA vs control siRNA). Tumor angiogenesis was quantified (DiI-perfusion and LASER confocal microscopy). EPC homing to tumor vasculature was detected by immunostaining. Explanted in vivo tumor size and weight were measured. Results:Three melanoma cells tested expressed undetectable levels of SDF-1&agr;. Additional enforced overexpression of SDF-1&agr; (by Lenti-SDF-1&agr;) increased melanoma cell growth both in vitro and in vivo, enhanced EPC homing to tumor tissue, and increased tumor angiogenesis. Knocking-down vascular E-selectin significantly inhibited SDF-1&agr;-induced EPC homing, tumor angiogenesis, and decreased melanoma growth in vivo. Conclusions:Downregulation of vascular E-selectin profoundly inhibits EPC homing, tumor angiogenesis, and tumor growth in human melanoma xenograft murine model, potentially by suppression of E-selectin-mediated EPC-endothelial cells interactions/homing. These findings identify E-selectin as a novel target for inhibition of melanoma angiogenesis and tumor growth.

Directing and Potentiating Stem Cell-Mediated Angiogenesis and Tissue Repair by Cell Surface E-Selectin Coating.

Stem cell therapy has emerged as a promising approach for treatment of a number of diseases, including delayed and non-healing wounds. However, targeted systemic delivery of therapeutic cells to the dysfunctional tissues remains one formidable challenge. Herein, we present a targeted nanocarrier-mediated cell delivery method by coating the surface of the cell to be delivered with dendrimer nanocarriers modified with adhesion molecules. Infused nanocarrier-coated cells reach to destination via recognition and association with the counterpart adhesion molecules highly or selectively expressed on the activated endothelium in diseased tissues. Once anchored on the activated endothelium, nanocarriers-coated transporting cells undergo transendothelial migration, extravasation and homing to the targeted tissues to execute their therapeutic role. We now demonstrate feasibility, efficacy and safety of our targeted nanocarrier for delivery of bone marrow cells (BMC) to cutaneous wound tissues and grafted corneas and its advantages over conventional BMC transplantation in mouse models for wound healing and neovascularization. This versatile platform is suited for targeted systemic delivery of virtually any type of therapeutic cell.

SDF-1α-induced dual pairs of E-selectin/ligand mediate endothelial progenitor cell homing to critical ischemia

Homing of endothelial progenitor cells (EPC) to the ischemic tissues is a key event in neovascularization and tissue regeneration. In response to ischemic insult, injured tissues secrete several chemo-cytokines, including stromal cell-derived factor-1α (SDF-1α), which triggers mobilization and homing of bone marrow-derived EPC (BMD-EPC). We previously reported that SDF-1α-induced EPC homing is mediated by a panel of adhesion molecules highly or selectively expressed on the activated endothelium in ischemic tissues, including E-selectin. Elevated E-selectin on wound vasculature serve as docking sites for circulating EPC, which express counterpart E-selectin ligands. Here, we show that SDF-1α presented in wound tissue and released into circulation can act both locally and remotely to induce ischemic tissue endothelium and BMD-EPC to express both E-selectin and its ligands. By performing BM transplantation using E-selectin−/− and E-selectin+/+ mice as the donors and recipients respectively, we demonstrate that upregulated dual E-selectin/ligand pairs reciprocally expressed on ischemic tissue endothelium and BMD-EPC act as double-locks to secure targeted EPC- endothelium interactions by which to facilitate EPC homing and promote neovascularization and tissue repair. These findings describe a novel mechanism for BMD-EPC homing and indicate that dual E-selectin/ligand pairs may be effective targets/tools for therapeutic neovascularization and targeted cell delivery.

Oral nicotine aggravates endothelial dysfunction and vascular inflammation in diet-induced obese rats: Role of macrophage TNFα

Obesity and cigarette smoke are major cardiovascular (CV) risk factors and, when coexisting in the same individuals, have additive/synergistic effects upon CVD. We studied the mechanisms involved in nicotine enhancement of CVD in Sprague Dawley rats with diet–induced obesity. The rats were fed either a high fat (HFD) or normal rat chow diet with or without nicotine (100 mg/L in drinking water) for 20 weeks. HFD rats developed central obesity, increased systolic blood pressure (SBP), aortic superoxide (O2-) production, and impaired endothelial nitric oxide synthase (eNOS) and endothelium-dependent relaxation to acetylcholine (EDR). Nicotine further increased SBP, O2- and impaired eNOS and EDR in obese rats. In the peritoneal macrophages from obese rats, tumor necrosis factor (TNF) α, interleukin 1β and CD36 were increased, and were further increased in nicotine-treated obese rats. Using PCR array we found that 3 of 84 target proinflammatory genes were increased by 2–4 fold in the aorta of obese rats, 11 of the target genes were further increased in nicotine-treated obese rats. HUVECs, incubated with conditioned medium from the peritoneal macrophages of nicotine treated-obese rats, exhibited reduced eNOS and increased NADPH oxidase subunits gp91phox and p22phox expression. Those effects were partially prevented by adding anti-TNFα antibody to the conditioned medium. Our results suggest that nicotine aggravates the CV effects of diet–induced obesity including the oxidative stress, vascular inflammation and endothelial dysfunction. The underlying mechanisms may involve in targeting endothelium by enhancement of macrophage-derived TNFα.

The effect of estrogen on diabetic wound healing is mediated through increasing the function of various bone marrow-derived progenitor cells

Objective: Endothelial progenitor cells (EPCs) are the key cells of postnatal neovascularization, and mesenchymal stem cells (MSCs) possess pluripotent differentiation capacity and contribute to tissue regeneration and wound healing. Both EPCs and MSCs are critical to the wound repair process, which is hindered in diabetes mellitus. Diabetes has been shown to decrease the function of these progenitor cells, whereas estrogen has beneficial wound healing effects. However, the role of estrogen in modulating EPC and MSC biology in diabetes is unknown. We investigated the effect of estrogen on improving bone marrow (BM)‐derived EPC and MSC function using a murine diabetic wound healing model. Methods: Female diabetic db+/db+ and nondiabetic control mice were wounded cutaneously and treated with topical estrogen or placebo cream. On day 5 after wounding, BM cells were harvested to quantify EPC number and colony‐forming units of EPCs and MSCs. Wound healing rate was concurrently studied. Vessel density and scar density were then quantified using whole body perfusion and laser confocal microscopy. EPC recruitment was documented by immunohistochemistry to identify CD34‐ and vascular endothelial growth factor receptor 2‐positive cells in the vessel wall. Data were analyzed by analysis of variance. Results: Topical estrogen significantly increased colony‐forming units of both EPCs and MSCs compared with placebo treatment, indicating improved viability and proliferative ability of these cells. Consistently, increased recruitment of EPCs to diabetic wounds and higher vessel density were observed in estrogen‐treated compared with placebo‐treated mice. Consequently, topical estrogen significantly accelerated wound healing as early as day 6 after wounding. In addition, scar density resulting from collagen deposition was increased in the estrogen‐treated group, reflecting increased MSC activity and differentiation. Conclusions: Estrogen treatment increases wound healing and wound neovascularization in diabetic mice. Our data implicate that these beneficial effects may be mediated through improving the function of BM‐derived EPCs and MSCs. Clinical Relevance: This study reveals a novel mechanism for the beneficial effect of topical estrogen treatment on the improvement of diabetic wound healing.