Engineering of implantable tissues requires rapid induction of angiogenesis to meet the significant oxygen and nutrient demands of cells during tissue repair. To this end, our laboratories have utilized medicinal chemistry to synthesize non-peptide-based inducers of angiogenesis to aid tissue engineering. In this study, we describe the evaluation of SC-3-149, a small molecule compound with proliferative effects on vascular endothelial cells. Specifically, exogenous exposure of SC-3-149 induced an 18-fold increase in proliferation of human microvascular endothelial cells in vitro at low micromolar potency by day 14 in culture. Moreover, SC-3-149 significantly increased the formation of endothelial cord and tubelike structures in vitro, and improved endothelial scratch wound healing within 24 h. SC-3-149 also significantly inhibited vascular endothelial cell death owing to serum deprivation and high acidity (pH 6). Concurrent incubation of SC-3-149 with vascular endothelial growth factor increased cell survivability under serum-deprived conditions by an additional 7%. In addition, in vivo injection of SC-3-149 into the rat mesentery produced qualitative increases in microvessel length density. Taken together, our studies suggest that SC-3-149 and its analogs may serve as promising new angiogenic agents for targeted drug delivery and therapeutic angiogenesis in tissue engineering.
"Angiogenesis is the process of developing vasculature, which contributes to the growth of tumors and promotes metastasis (Reinhart-King 2008; Tonini et al. 2003). During the process of angiogenesis, endothelial cells acquire distinctive molecular signatures , and transform into an angiogenic phenotype, incurring various other changes in cellular behavior and function (Wieghaus et al. 2006). Vascular endothelial growth factors (VEGFs), epidermal growth factors (EGFs), platelet-derived endothelial cell growth factors (PDGFs), and acidic and basic fibroblast growth factors (aFGFs and bFGFs) are major angiogenic mediators as well as strong vascular permeability factors (Chiba et al. 2008; Li et al. 2005). "
[Show abstract][Hide abstract] ABSTRACT: This study examined the in vitro and in vivo angiogenic effects of norcantharidin (NCTD), a synthetic, small-molecule antitumor compound.
Syngeneic colorectal adenocarcinoma CT26 cells were implanted in mice to examine the effect of NCTD on VEGF production and renal and hepatic toxicity. Human umbilical endothelial cells (HUVECs) were used to examine the in vitro effect of NCTD on viability, chemotaxis, vascular network tube formation, adhesive ability, anoikis, and mitogen-activated protein kinase (MAPK) signaling. A protein array was used for analysis of angiogenic factors released from NCTD-treated HUVECs.
NCTD suppressed plasma VEGF levels of tumor-bearing mice, without renal or hepatic toxicity. In vitro, NCTD inhibited viability of normal HUVECs to a lesser extent than CT26 cancer cells. At concentrations less than those inhibiting 50% of the cells, NCTD inhibited migration and capillary-like tube formation of HUVECs. The anti-angiogenic effect of NCTD was accompanied by anoikis, down-regulation of integrin beta1, and breakdown of vimentin. NCTD decreased MAPK expression of phosphorylated (p)-JNK and p-ERK. P-P38 expression or P38 inhibitor SB203580 did not impair the effect of NCTD on viability or adhesion of HUVECs. In addition, NCTD inhibited the release of pro-angiogenic factors from HUVECs, but not from CT26 cells.
NCTD is a synthetic, small-molecule compound possessing anti-angiogenetic activity with potential use in anti-cancer therapy as an anti-metastatic and anti-angiogenic agent.
Life sciences 09/2009; 85(17-18):642-51. DOI:10.1016/j.lfs.2009.09.003 · 2.70 Impact Factor
"Panel (a) illustrates representative images of HMVECs stimulated with vehicle control, PNF1, PNF1 + TIMP-1, or PNF1 + TIMP-2 (scale bar = 50 μm). In accordance with previous experiments performed in Matrigel (see (Wieghaus et al. 2006)), PNF1 stimulated more cell elongation and more cell-cell associations than vehicle control samples. This activity was attenuated in response to 2.5 μg/mL TIMP-2, an inhibitor of MT1-MMP, but not in response to 2.5 μg/mL TIMP-1, an inhibitor of soluble MMPs but not membrane-bound MT1-MMP (Sodek et al. 2007). "
[Show abstract][Hide abstract] ABSTRACT: We are creating synthetic pharmaceuticals with angiogenic activity and potential to promote vascular invasion. We previously demonstrated that one of these molecules, phthalimide neovascular factor 1 (PNF1), significantly expands microvascular networks in vivo following sustained release from poly(lactic-co-glycolic acid) (PLAGA) films. In addition, to probe PNF1 mode of action, we recently applied a novel pathway-based compendium analysis to a multi-timepoint, controlled microarray data set of PNF1-treated (vs. control) human microvascular endothelial cells (HMVECs), and we identified induction of tumor necrosis factor-alpha (TNF-alpha) and, subsequently, transforming growth factor-beta (TGF-beta) signaling networks by PNF1. Here we validate this microarray data set with quantitative real-time polymerase chain reaction (RT-PCR) analysis. Subsequently, we probe this data set and identify three specific TGF-beta-induced genes with regulation by PNF1 conserved over multiple timepoints-amyloid beta (A4) precursor protein (APP), early growth response 1 (EGR-1), and matrix metalloproteinase 14 (MMP14 or MT1-MMP)-that are also implicated in angiogenesis. We further focus on MMP14 given its unique role in angiogenesis, and we validate MT1-MMP modulation by PNF1 with an in vitro fluorescence assay that demonstrates the direct effects that PNF1 exerts on functional metalloproteinase activity. We also utilize endothelial cord formation in collagen gels to show that PNF1-induced stimulation of endothelial cord network formation in vitro is in some way MT1-MMP-dependent. Ultimately, this new network analysis of our transcriptional footprint characterizing PNF1 activity 1-48 h post-supplementation in HMVECs coupled with corresponding validating experiments suggests a key set of a few specific targets that are involved in PNF1 mode of action and important for successful promotion of the neovascularization that we have observed by the drug in vivo.
Biotechnology and Bioengineering 07/2009; 103(4):796-807. DOI:10.1002/bit.22310 · 4.13 Impact Factor
"Here, we developed and applied a novel pathway-based compendium analysis to our 1–48 h transcriptional regulatory footprint of PNF1 activity in HMVEC to specifically identify the molecular mechanism by which the drug functions. We demonstrate through this compendium analysis that PNF1 stimulation in its most effective in vitro concentration (30 µM) (Wieghaus et al., 2006) produces a significant change in expression of HMVEC genetic networks and related cell signaling processes, particularly TNF-α and TGF-β pathways. These changes are highly consistent with pro-angiogenic activity. "
[Show abstract][Hide abstract] ABSTRACT: MOTIVATION: Computational techniques have been applied to experimental datasets to identify drug mode-of-action. A shortcoming of existing approaches is the requirement of large reference databases of compound expression profiles. Here, we developed a new pathway-based compendium analysis that couples multi-timepoint, controlled microarray data for a single compound with systems-based network analysis to elucidate drug mechanism more efficiently. RESULTS: We applied this approach to a transcriptional regulatory footprint of phthalimide neovascular factor 1 (PNF1)-a novel synthetic small molecule that exhibits significant in vitro endothelial potency-spanning 1-48 h post-supplementation in human micro-vascular endothelial cells (HMVEC) to comprehensively interrogate PNF1 effects. We concluded that PNF1 first induces tumor necrosis factor-alpha (TNF-alpha) signaling pathway function which in turn affects transforming growth factor-beta (TGF-beta) signaling. These results are consistent with our previous observations of PNF1-directed TGF-beta signaling at 24 h, including differential regulation of TGF-beta-induced matrix metalloproteinase 14 (MMP14/MT1-MMP) which is implicated in angiogenesis. Ultimately, we illustrate how our pathway-based compendium analysis more efficiently generates hypotheses for compound mechanism than existing techniques.
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