Hongwei Shao

Statin and Stromal Cell-Derived Factor-1 Additively Promote Angiogenesis by Enhancement of Progenitor Cells Incorporation into New Vessels

Angiogenesis requires the mobilization of progenitor cells from the bone marrow and homing of progenitor cells to ischemic tissue. Statins facilitate the former, and the chemokine stromal cell‐derived factor‐1 (SDF‐1) enhances the latter. Their combined influence on angiogenesis was studied in vivo in the ischemic hindlimb C57BL/6 mouse model. The ischemic to non‐ischemic perfusion ratio increased from 0.29 ± 0.02 immediately after femoral excision to 0.51 ± 0.10 three weeks after the surgery in the mice treated with either fluvastatin or SDF‐1 alone, which is significantly better than the control (0.38 ± 0.05, p < .05, n = 6). The combined use of fluvastatin and SDF‐1 further improved the reperfusion ratio (0.62 ± 0.08, p < .05). More cell proliferation, less apoptosis, enhanced bone marrow‐derived endothelial progenitor cell (EPC) incorporation and higher capillary density were observed in ischemic tissue treated with both statin and SDF‐1. In vitro mono‐treatment with either fluvastatin (100 nM) or SDF‐1 (100 ng/ml) facilitated EPC proliferation and migration, inhibited EPC apoptosis, enhanced expression of matrix metalloproteinase‐2 (MMP‐2) and ‐9 (MMP‐9), and increased Akt phosphorylation and nitric oxide production. These effects were significantly augmented by the two agents together and ablated by inhibitors of either Akt or nitric oxide synthase (NOS). In conclusion, statin and SDF‐1 additively enhance progenitor cell migration and proliferation and down‐regulate EPC apoptosis, resulting in improved reperfusion via activation of the Akt/NOS pathway and up‐regulation of MMP‐2 and MMP‐9 expression.

Extracellular calcium increases CXCR4 expression on bone marrow-derived cells and enhances pro-angiogenesis therapy

Cell surface receptors play major roles in the mobilization and homing of progenitor cells from the bone marrow to peripheral tissues. CXCR4 is an important receptor that regulates homing of leucocytes and endothelial progenitors in response to the chemokine stromal cell‐derived factor‐1 (SDF‐1). Ionic calcium is also known to regulate chemotaxis of selective bone marrow cells (BMCs) through the calcium‐sensing receptor, CaR. Here we show that calcium regulates CXCR4 expression and BMC responses to SDF‐1. CaCl2 treatment of BMC induced a time‐ and dose‐dependent increase in both the transcription and cell surface expression of CXCR4. BMC subpopulations expressing VEGFR2+, CD34+ and cKit+/Sca‐1+ were especially sensitive to calcium. The effects were blocked by calcium influx inhibitors, anti‐CaR antibody and the protein synthesis inhibitor cycloheximide, but not by the CXCR4 antagonist AMD3100. Calcium treatment also enhanced SDF‐1‐mediated CXCR4 internalization. These changes were reflected in significantly improved chemotaxis by SDF‐1, which was abolished by AMD3100 and by antibody against CXCR4. Calcium pre‐treatment improved homing of CD34+ BMCs to ischemic muscle in vivo, and enhanced revascularization in ischemic mouse hindlimbs. Our results identify calcium as a positive regulator of CXCR4 expression that promotes stem cell mobilization, homing and therapy.

A novel CXCR4 antagonist derived from human SDF-1β enhances angiogenesis in ischaemic mice

AIMS The effects on angiogenesis of a novel CXC chemokine receptor 4 (CXCR4) antagonist, SDF-1betaP2G, derived from human stromal cell-derived factor-1beta (SDF-1beta), were examined in a model of hind limb ischaemia in mice. METHODS AND RESULTS The antagonistic activities of SDF-1betaP2G against CXCR4 were evaluated in vitro and in vivo and compared with phosphate-buffered saline and AMD3100 (a small bicyclam antagonist of SDF-1). Angiogenesis, muscle regeneration and the expression of pro-angiogenic factors were evaluated in ischaemic gastrocnemius muscles. Distant toxic effects of SDF-1betaP2G were evaluated by inflammatory and apoptotic markers. SDF-1betaP2G induced CXCR4 internalization and competitively inhibited the chemotaxis of SDF-1beta but did not mediate migration, calcium influx, or the phosphorylation of Akt and extracellular signal-regulated kinase in cultured T-lymphoblastic leukaemia cells or H9C2 cells. SDF-1betaP2G enhanced blood flow, angiogenesis, and muscle regeneration in ischaemic hind limbs, and the enhancement was significantly better than that of AMD3100. Markers of angiogenesis and progenitor cell migration, including phosphorylated Akt, vascular endothelial growth factor (VEGF), SDF-1 and CXCR4, were up-regulated by SDF-1betaP2G and co-localized with CD31-positive cells. Neutralization of VEGF with its specific antibody abolished SDF-1betaP2G-induced blood reperfusion and angiogenesis. No apparent inflammatory and apoptotic effects were found in heart, liver, kidneys, and testes after SDF-1betaP2G administration. CONCLUSION Our findings indicate that the novel CXCR4 antagonist, SDF-1betaP2G, can efficiently enhance ischaemic angiogenesis, blood flow restoration, and muscle regeneration without apparent adverse effects, most likely through a VEGF-dependent pathway.

Activation of Notch1 signaling in stromal fibroblasts inhibits melanoma growth by upregulating WISP-1

The tumor microenvironment is emerging as an important target for cancer therapy. Fibroblasts (Fbs) within the tumor stroma are critically involved in promoting tumor growth and angiogenesis through secretion of soluble factors, synthesis of extracellular matrix and direct cell–cell interaction. In this work, we aim to alter the biological activity of stromal Fbs by modulating the Notch1 signaling pathway. We show that Fbs engineered to constitutively activate the Notch1 pathway significantly inhibit melanoma growth and tumor angiogenesis. We determine that the inhibitory effect of ‘Notch-engineered’ Fbs is mediated by increased secretion of Wnt-induced secreted protein-1 (WISP-1) as the effects of Notch1 activation in Fbs are reversed by shRNA-mediated blockade of WISP-1. When ‘Notch-engineered’ Fbs are co-grafted with melanoma cells in SCID mice, shRNA-mediated blockade of WISP-1 reverses the tumor-suppressive phenotype of the ‘Notch-engineered’ Fbs, significantly increases melanoma growth and tumor angiogenesis. Consistent with these findings, supplement of recombinant WISP-1 protein inhibits melanoma cell growth in vitro. In addition, WISP-1 is modestly expressed in melanoma-activated Fbs but highly expressed in inactivated Fbs. Evaluation of human melanoma skin biopsies indicates that expression of WISP-1 is significantly lower in melanoma nests and surrounding areas filled with infiltrated immune cells than in the adjacent dermis unaffected by the melanoma. Overall, our study shows that constitutive activation of the Notch1 pathway confers Fbs with a suppressive phenotype to melanoma growth, partially through WISP-1. Thus, targeting tumor stromal Fbs by activating Notch signaling and/or increasing WISP-1 may represent a novel therapeutic approach to combat melanoma.

Targeting Notch Signaling for Cancer Therapeutic Intervention

The Notch signaling pathway is an evolutionarily conserved, intercellular signaling cascade. The Notch proteins are single-pass receptors that are activated upon interaction with the Delta (or Delta-like) and Jagged/Serrate families of membrane-bound ligands. Association of ligand-receptor leads to proteolytic cleavages that liberate the Notch intracellular domain (NICD) from the plasma membrane. The NICD translocates to the nucleus, where it forms a complex with the DNA-binding protein CSL, displacing a histone deacetylase (HDAc)-corepressor (CoR) complex from CSL. Components of a transcriptional complex, such as MAML1 and histone acetyltransferases (HATs), are recruited to the NICD-CSL complex, leading to the transcriptional activation of Notch target genes. The Notch signaling pathway plays a critical role in cell fate decision, tissue patterning, morphogenesis, and is hence regarded as a developmental pathway. However, if this pathway goes awry, it contributes to cellular transformation and tumorigenesis. There is mounting evidence that this pathway is dysregulated in a variety of malignancies, and can behave as either an oncogene or a tumor suppressor depending upon cell context. This chapter highlights the current evidence for aberration of the Notch signaling pathway in a wide range of tumors from hematological cancers, such as leukemia and lymphoma, through to lung, skin, breast, pancreas, colon, prostate, ovarian, brain, and liver tumors. It proposes that the Notch signaling pathway may represent novel target for cancer therapeutic intervention.

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.

Defective CXCR4 expression in aged bone marrow cells impairs vascular regeneration.

The chemokine stromal cell‐derived factor‐1 (SDF‐1) plays a critical role in mobilizing precursor cells in the bone marrow and is essential for efficient vascular regeneration and repair. We recently reported that calcium augments the expression of chemokine receptor CXCR4 and enhances the angiogenic potential of bone marrow derived cells (BMCs). Neovascularization is impaired by aging therefore we suggested that aging may cause defects of CXCR4 expression and cellular responses to calcium. Indeed we found that both the basal and calcium‐induced surface expression of CXCR4 on BMCs was significantly reduced in 25‐month‐old mice compared with 2‐month‐old mice. Reduced Ca‐induced CXCR4 expression in BMC from aged mice was associated with defective calcium influx. Diminished CXCR4 surface expression in BMC from aged mice correlated with diminished neovascularization in an ischemic hindlimb model with less accumulation of CD34+ progenitor cells in the ischemic muscle with or without local overexpression of SDF‐1. Intravenous injection of BMCs from old mice homed less efficiently to ischemic muscle and stimulated significantly less neovascularization compared with the BMCs from young mice. Transplantation of old BMCs into young mice did not reconstitute CXCR4 functions suggesting that the defects were not reversible by changing the environment. We conclude that defects of basal and calcium‐regulated functions of the CXCR4/SDF‐1 axis in BMCs contribute significantly to the age‐related loss of vasculogenic responses.

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.

Blood-derived smooth muscle cells as a target for gene delivery

OBJECTIVE To examine the feasibility of using blood-derived smooth muscle cells (BD-SMCs) as a target for to deliver therapeutic proteins. MATERIALS AND METHODS Mononuclear cells (MNC) were isolated from peripheral blood. The outgrowth colonies from MNC culture were differentiated into BD-SMCs in media containing platelet-derived growth factor BB. Phenotypic characterization of BD-SMCs was assessed by immunocytochemistry. Cell proliferation, gene transfer efficiency with a retroviral vector, apoptosis, and the biological activity of the transduced gene product from the BD-SMCs were evaluated in vitro and in vivo in comparison with vascular derived SMC (VSMCs). RESULTS BD-SMCs stained positive for SMC markers. No significant difference was observed between BD-SMCs and VSMCs in cell proliferation, migration, adhesiveness, and gene transfer efficiency. After BD-SMCs were transduced with a retroviral vector carrying the secreted alkaline phosphatase gene (SEAP), 174 +/- 50 mug biologically active SEAP was produced per 10(6) cells over 24 hours. After injecting 5 x 10(6) cells expressing SEAP intravenously into rabbits, SEAP concentration increased significantly in the circulation from 0.14 +/- 0.04 mug/ml to 2.34 +/- 0.16 mug/ml 3 days after cell injection (P < .01, n = 3). Circulating levels of SEAP decreased to 1.76 mug /ml 1 week later and remained at this level up to 8 weeks, then declined to pre-cell injection level at 12 weeks. VSMC in vivo gene expression data were equivalent. CONCLUSION BD-SMCs have similar characteristics to mature VSMCs and can be used as a novel target for gene transfer to deliver a therapeutic protein.

Abstract 5010: Activation of the notch signaling pathway confers a tumor-suppressive phenotype on melanoma-associated fibroblasts

Objectives. The tumor microenvironment (TME) is an emerging therapeutic target for cancer treatment. Cancer-associated fibroblasts (CAF) play a crucial role in cancer progression. We aim to target TME by altering intracellular signaling which determines the biological function of CAF. We have recently showed that the Notch signaling pathway likely functions as a molecular switch in controlling the tumor regulatory role of CAF in animal models and experimentally created “CAF”. Here, we investigated the status of Notch signaling in human melanoma-associated fibroblasts (MAF) versus their normal counterparts and tested whether manipulation of the Notch pathway activity in MAF alter their tumor-regulating function. Methods. We examined levels of Hes1, a canonical Notch target, in MAF of human malignant melanoma at different stages (I-IV) and fibroblasts in either adjacent or non-adjacent normal skin tissues using tissue microarray. MAF were isolated from human metastatic melanoma tissues. Notch pathway RT2-PCRArray and immunoblot were used to assess Notch pathway activity in MAF versus normal human dermal fibroblasts. Activation of Notch signaling pathway in MAF was achieved by lentiviral vector encoding active form of Notch1 (NIC). The effect of Notch1-engineered MAF on melanoma growth was tested by in vitro co-cultures and in a mouse co-xenograft model (n=6/group). Tumor angiogenesis was analyzed by immunochemistry. Results. MAF expressed decreased levels of Hes1 compared with adjacent skin fibroblasts. Isolated MAF also exhibited lower Notch activity than normal human dermal fibroblasts. Notch1-engineered MAF significantly inhibited melanoma cell growth in vitro (p Conclusions. Notch pathway activity is down-regulated in MAF. Increase of Notch pathway activity confers MAF with inhibition to melanoma growth and tumor angiogenesis. Our study demonstrates that Notch signaling is a critical molecular switch in determining the tumor regulatory role of MAF and provides potential targets for cancer therapeutic interventions on the TME. Citation Format: Hongwei Shao, Mecker G. Moller, Long Cai, Leiming Zhang, Zhao-Jun Liu. Activation of the notch signaling pathway confers a tumor-suppressive phenotype on melanoma-associated fibroblasts [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5010. doi:10.1158/1538-7445.AM2017-5010