Neovascularization is essential for the survival and successful integration of most engineering tissues after implantation in vivo. The objective of this study was to elucidate possible mechanisms of phthalimide neovascular factor 1 (PNF1), a new synthetic small molecule proposed for therapeutic induction of angiogenesis. Complementary deoxyribonucleic acid microarray analysis was used to identify 568 transcripts in human microvascular endothelial cells (HMVECs) that were significantly regulated after 24-h stimulation with 30 μM of PNF1, previously known as SC-3–149. Network analysis tools were used to identify genetic networks of the global biological processes involved in PNF1 stimulation and to describe known molecular and cellular functions that the drug regulated most highly. Examination of the most significantly perturbed networks identified gene products associated with transforming growth factor-beta (TGF-β), which has many known effects on angiogenesis, and related signal transduction pathways. These include molecules integral to the thrombospondin, plasminogen, fibroblast growth factor, epidermal growth factor, ephrin, Rho, and Ras signaling pathways that are essential to endothelial function. Moreover, real-time reverse-transcriptase polymerase chain reaction (RT-PCR) of select genes showed significant increases in TGF-β-associated receptors endoglin and beta glycan. These experiments provide important insight into the pro-angiogenic mechanism of PNF1, namely, TGF-β-associated signaling pathways, and may ultimately offer new molecular targets for directed drug discovery.

Poly(D,L-lactic-co-glycolic acid) (PLGA) is a biodegradable polymer that is widely used for drug delivery. However, the degradation of PLGA alters the local microenvironment and may influence tissue structure and/or function. Here, we studied whether PLGA degradation affects the structure of the arteriolar microcirculation through arteriogenic expansion of maximum lumenal diameters and/or the formation of new smooth muscle-coated vessels. Single microspheres comprised of 50:50 PLGA (521 ± 52.7 μm diameter), 50:50 PLGA with bovine serum albumin (BSA) (547 ± 62.2 μm), 85:15 PLGA (474 ± 52.6 μm), or 85:15 PLGA with BSA (469 ± 57.2 μm) were implanted into mouse dorsal skinfold window chambers, and longitudinal arteriolar diameter measurements were made in the presence of a vasodilator (10−4M adenosine) over 7 days. At the end of the 7-day period, the length density of all smooth muscle-coated microvessels was also determined. Implantation of the window chamber alone elicited a 22% increase in maximum arteriolar diameter. However, the addition of 85:15 and 50:50 PLGA microspheres, bearing either BSA or no protein, elicited a significant enhancement of this arteriogenic response, with final maximum arteriolar diameters ranging from 36 to 46% more than their original size. Interestingly, the influence of PLGA degradation on microvascular structure was limited to lumenal arteriolar expansion, as we observed no significant differences in length density of smooth muscle-coated microvessels. We conclude that the degradation of PLGA microspheres may elicit an arteriogenic response in subcutanteous tissue in the dorsal skinfold window chamber; however, it has no apparent effect on the total length of smooth muscle-coated microvasculature.

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 dataset of PNF1-treated (versus control) human microvascular endothelial cells (HMVECs), and we identified induction of tumor necrosis factor-alpha (TNF-α) and, subsequently, transforming growth factor-beta (TGF-β) 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 dataset and identify three specific TGF-β-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.

Phthalimide neovascular factor (PNF1, formerly SC-3-149) is a potent stimulator of proangiogenic signaling pathways in endothelial cells. In this study, we evaluated the in vivo effects of sustained PNF1 release to promote ingrowth and expansion of microvascular networks surrounding biomaterial implants. The dorsal skinfold window chamber was used to evaluate the structural remodeling response of the local microvasculature. PNF1 was released from poly(lactic-co-glycolic acid) (PLAGA) films, and a transport model was utilized to predict PNF1 penetration into the surrounding tissue. PNF1 significantly expanded microvascular networks within a 2 mm radius from implants after 3 and 7 days by increasing microvessel length density and lumenal diameter of local arterioles and venules. Staining of histological sections with CD11b showed enhanced recruitment of circulating white blood cells, including monocytes, which are critical for the process of vessel enlargement through arteriogenesis. As PNF1 has been shown to modulate MT1-MMP, a facilitator of CCL2 dependent leukocyte transmigration, aspects of window chamber experiments were repeated in CCR2−/− (CCL2 receptor) mouse chimeras to more fully explore the critical nature of monocyte recruitment on the therapeutic benefits of PNF1 function in vivo.

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-α) signaling pathway function which in turn affects transforming growth factor-beta (TGF-β) signaling. These results are consistent with our previous observations of PNF1-directed TGF-β signaling at 24 h, including differential regulation of TGF-β-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.

Availability: The microarray data generated as part of this study are available in the Gene Expression Omnibus (http://www.ncbi.nlm.nih.gov/geo/).

Contact: botchwey@virginia.edu; papin@virginia.edu

Supplementary information: Supplementary data are available at Bioinformatics online.

Sphingosine 1-phosphate (S1P) is a bioactive phospholipid that impacts migration, proliferation, and survival in diverse cells types, including endothelial cells, smooth muscle cells, and osteoblast-like cells. In this study, we investigated the effects of sustained release of S1P on microvascular remodeling and associated bone defect healing in vivo. The murine dorsal skinfold window chamber model was used to evaluate the structural remodeling response of the microvasculature. Our results demonstrated that 1:400 (w/w) loading and subsequent sustained release of S1P from poly(lactic-co-glycolic acid) (PLAGA) significantly enhanced lumenal diameter expansion of arterioles and venules after 3 and 7 days. Incorporation of 5-bromo-2-deoxyuridine (BrdU) at day 7 revealed significant increases in mural cell proliferation in response to S1P delivery. Additionally, three-dimensional (3D) scaffolds loaded with S1P (1:400) were implanted into critical-size rat calvarial defects and healing of bony defects was assessed by radiograph x-ray, microcomputed tomography (μCT), and histology. Sustained release of S1P significantly increased the formation of new bone after 2 and 6 weeks of healing and histological results suggest increased numbers of blood vessels in the defect site. Taken together, these experiments support the use of S1P delivery for promoting microvessel diameter expansion and improving the healing outcomes of tissue-engineered therapies.