Pro-angiogenic Gene Expression Research Articles

Hedgehog signaling is critical for maintenance of the adult coronary vasculature in mice

Hedgehog (HH) signaling has emerged as a critical pathway involved in the pathogenesis of a variety of tumors. As a result, HH antagonists are currently being evaluated as potential anticancer therapeutics. Conversely, activation of HH signaling in the adult heart may be beneficial, as HH agonists have been shown to increase coronary vessel density and improve coronary function after myocardial infarction. To investigate a potential homeostatic role for HH signaling in the adult heart, we ablated endogenous HH signaling in murine myocardial and perivascular smooth muscle cells. HH signaling was required for proangiogenic gene expression and maintenance of the adult coronary vasculature in mice. In the absence of HH signaling, loss of coronary blood vessels led to tissue hypoxia, cardiomyocyte cell death, heart failure, and subsequent lethality. We further showed that HH signaling specifically controlled the survival of small coronary arteries and capillaries. Together, these data demonstrate that HH signaling is essential for cardiac function at the level of the coronary vasculature and caution against the use of HH antagonists in patients with prior or ongoing heart disease.

Angiotensin Converting Enzyme Inhibition Enhances Collateral Artery Remodeling in Rats With Femoral Artery Occlusion

Evidence from experimental animal studies indicate that ACE inhibition expands collateral blood flow both in ischemic hearts and peripheral limbs. The present study evaluates whether ACE inhibitor induces collateral blood flow expansion and change of angiogenic gene expression profile in collateral arteries during remodeling. Male Sprague-Dawley rats, weighing 350 g were treated with vehicle (control) or quinapril (ACE inhibitor) at either low dose (3.0 mg/kg) or high dose (18 mg/kg) for 1, 3, 7, 14 days (gene expression) or 16 days (blood flow). All rats received bilateral occlusions of the femoral arteries. Collateral blood flow to the hind limb was assessed by 85Sr and 141Ce-labeled microspheres during treadmill running at 15 and 25 m/min speeds. Quinapril reduced plasma ACE activity by 58% and 88% for the low-dose and high-dose groups, respectively (P < 0.001). High-dose quinapril reduced exercising blood pressure (P < 0.05) and increased hind limb conductance. Collateral blood flows to calf muscles were 51 +/- 3.7, 73 +/- 5.0, and 68 +/- 1.9 mL/min per 100 g in control and quinapril low- and high-dose groups, respectively, during high-speed running (P < 0.001). Real-time RT-PCR revealed that ACE inhibition shifted gene expression to a proangiogenic phenotype in the newly developed collateral arteries. Our findings indicate that ACE inhibition could increase collateral-dependent blood flow and collateral vessel remodeling by promoting proangiogenic gene expression in newly developed collateral arteries. Our results support the potential utility of ACE inhibitor as a therapeutic agent in treating peripheral occlusive arterial disease.

Oncolytic HSV and Erlotinib Inhibit Tumor Growth and Angiogenesis in a Novel Malignant Peripheral Nerve Sheath Tumor Xenograft Model

Malignant peripheral nerve sheath tumors (MPNSTs), driven in part by hyperactive Ras and epidermal growth factor receptor (EGFR) signaling, are often incurable. Testing of therapeutics for MPNST has been hampered by lack of adequate xenograft models. We previously documented that human MPNST cells are permissive for lytic infection by oncolytic herpes simplex viruses (oHSV). Herein we developed and characterized a xenograft model of human MPNST and evaluated the antitumor effects of oHSV mutants (G207 and hrR3) and the EGFR inhibitor, erlotinib. Additive cytotoxicity of these agents was found in human MPNST cell lines, suggesting that EGFR signaling is not critical for virus replication. Mice bearing human MPNST tumors treated with G207 or hrR3 by intraperitoneal or intratumoral injection showed tumor-selective virus biodistribution, virus replication, and reduced tumor burden. oHSV injection demonstrated more dramatic antitumor activity than erlotinib. Combination therapies showed a trend toward an increased antiproliferative effect. Both oHSV and erlotinib were antiangiogenic as measured by proangiogenic gene expression, effect on endothelial cells and xenograft vessel density. Overall, oHSVs showed highly potent antitumor effects against MPNST xenografts, an effect not diminished by EGFR inhibition. Our data suggest that inclusion of MPNSTs in clinical trials of oHSV is warranted.

SSeCKS/Gravin/AKAP12 attenuates expression of proliferative and angiogenic genes during suppression of v-Src-induced oncogenesis

BackgroundSSeCKS is a major protein kinase C substrate with kinase scaffolding and metastasis-suppressor activity whose expression is severely downregulated in Src- and Ras-transformed fibroblast and epithelial cells and in human prostate, breast, and gastric cancers. We previously used NIH3T3 cells with tetracycline-regulated SSeCKS expression plus a temperature-sensitive v-Src allele to show that SSeCKS re-expression inhibited parameters of v-Src-induced oncogenic growth without attenuating in vivo Src kinase activity.MethodsWe use cDNA microarrays and semi-quantitative RT-PCR analysis to identify changes in gene expression correlating with i) SSeCKS expression in the absence of v-Src activity, ii) activation of v-Src activity alone, and iii) SSeCKS re-expression in the presence of active v-Src.ResultsSSeCKS re-expression resulted in the attenuation of critical Src-induced proliferative and pro-angiogenic gene expression including Afp, Hif-1α, Cdc20a and Pdgfr-β, and conversely, SSeCKS induced several cell cycle regulatory genes such as Ptpn11, Gadd45a, Ptplad1, Cdkn2d (p19), and Rbbp7.ConclusionOur data provide further evidence that SSeCKS can suppress Src-induced oncogenesis by modulating gene expression downstream of Src kinase activity.

Bombesin stimulates nuclear factor kappa B activation and expression of proangiogenic factors in prostate cancer cells. Levine L, Lucci JA 3rd, Pazdrak B, Cheng JZ, Guo YS, Townsend CM Jr, Hellmich MR, Department of Obstetrics and Gynecology, The University of Texas Medical Branch, Galveston, TX 77555: Cancer Res 2003;63(13):3495–3502

The majority of deaths from prostate cancer occur in patients with androgen-insensitive metastatic disease. An important early event in the development of the metastatic phenotype is the induction of genes that promote angiogenesis, such as vascular endothelial growth factor (VEGF) and interleukin-8 (IL-8), which are released from tumor cells into their microenvironment. Coincident with progression from prostatic carcinoma in situ to metastatic disease is an increase in the number of tumor cells exhibiting neuroendocrine (NE) differentiation. NE cells express a variety of peptide hormones, including the bombesin (BBS)-like peptide, gastrin-releasing peptide (GRP), and its cognate receptor, GRP-R. Although there is a strong positive correlation between the degree of NE differentiation and the metastatic potential of prostate cancers, a mechanistic link between increased expression of peptide hormone receptors, such as GRP-R, and proangiogenic gene expression has not been established. Here we report that BBS stimulates nuclear factor kappa B (NF kappa B) activation and proangiogenic gene expression in the androgen-insensitive prostate cancer cells lines, PC-3 and DU-145. In PC-3 cells, BBS stimulation of GRP-R resulted in the up-regulation of IL-8 and VEGF expression through a NF kappa B-dependent pathway. We show that BBS treatment induced inhibitor of NF kappa B degradation, NF kappa B translocation to the cell nucleus, increased NF kappa B binding to its DNA consensus sequence, and increased IL-8 and VEGF mRNA expression and protein secretion. Treatment with the proteasome inhibitor, MG-132, blocked BBS-stimulated NF kappa B DNA binding, and IL-8 and VEGF expression and secretion. Finally, media collected from PC-3 cell cultures, after BBS treatment, stimulated an NF kappa B-dependent migration of human umbilical vascular endothelial cells in vitro. Together, our data demonstrate a role for BBS and GRP-R in the NF kappa B-dependent up-regulation of proangiogenic gene expression, and suggest a possible molecular mechanism linking NE differentiation and the increased metastatic potential of androgen-insensitive prostate cancers.

Endostatin gene transfer inhibits joint angiogenesis and pannus formation in inflammatory arthritis.

Rheumatoid arthritis is a prevalent example of an inflammatory angiogenic disease, which is mediated by pro-inflammatory and pro-angiogenic cytokines such as tumor necrosis factor (TNF). To evaluate the effect of the potent anti-angiogenic factor endostatin on TNF-induced inflammatory arthritis, we injected an endostatin-expressing lentiviral vector directly into the joints of human TNF-transgenic mice before the onset of disease. Histological analysis of the injected joints 8 weeks later revealed that endostatin reduced blood vessel density within the synovial tissues and an overall mean arthritis index. In vitro and in vivo examination of the potential mechanism by which endostatin inhibited the arthritis revealed that endostatin blocks TNF-induced activation of JNK and JNK-dependent pro-angiogenic gene expression. These data suggest a novel mechanism by which endostatin inhibits angiogenesis, and demonstrates the potential utility of anti-angiogenic gene therapy for treatment of inflammatory arthritis.