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Summary To gain insight into the cellular and molecular cues that promote neurovascular co-patterning at the earliest stages of human embryogenesis, we developed a human embryonic stem cell model to mimic the developing epiblast. Contact of ectoderm-derived neural cells with mesoderm-derived vasculature is initiated via the neural crest (NC), not the neural tube (NT). Neurovascular co-patterning then ensues with specification of NC toward an autonomic fate requiring vascular endothelial cell (EC)-secreted nitric oxide (NO) and direct contact with vascular smooth muscle cells (VSMCs) via T-cadherin-mediated homotypic interactions. Once a neurovascular template has been established, NT-derived central neurons then align themselves with the vasculature. Our findings reveal that, in early human development, the autonomic nervous system forms in response to distinct molecular cues from VSMCs and ECs, providing a model for how other developing lineages might coordinate their co-patterning., Graphical Abstract, Highlights • Neural crest (NC) cells drive neurovascular co-patterning, as modeled by hESC • Autonomic differentiation of NC cells depends on contact with perivascular cells • This requires endothelial-derived NO and T-cadherin-mediated interaction with VSMCs, Cheresh, Snyder, and colleagues demonstrate how direct contact between hESC-derived neural crest (NC) cells and the vasculature promotes autonomic differentiation. In a hESC model mimicking the developing epiblast, NC cells initiate neurovascular co-patterning prior to forming neural tube neurons. Endothelial-secreted NO and direct contact with vascular smooth muscle cells via T-cadherin are required to promote NC differentiation into autonomic neurons.

The ATR pathway is a critical mediator of the replication stress response in cells. In aberrantly proliferating cancer cells, this pathway can help maintain sufficient genomic integrity for cancer cell progression. Herein we describe the discovery of 19, a pyrazolopyrimidine-containing inhibitor of ATR via a strategic repurposing of compounds targeting PI3K.

While enzalutamide and abiraterone are approved for treatment of metastatic castration-resistant prostate cancer (mCRPC), approximately 20-40% of patients have no response to these agents. It has been stipulated that the lack of response and the development of secondary resistance to these drugs may be due to the presence of AR splice variants. HDAC6 has a role in regulating the androgen receptor (AR) by modulating heat shock protein 90 (Hsp90) acetylation, which controls the nuclear localization and activation of the AR in androgen-dependent and independent scenarios. With dual-acting AR-HDAC6 inhibitors it should be possible to target patients who don't respond to enzalutamide. Herein, we describe the design, synthesis and biological evaluation of dual-acting compounds which target AR and are also specific towards HDAC6. Our efforts led to compound 10 which was found to have potent dual activity (HDAC6 IC50=0.0356μM and AR binding IC50=

Although it is well established that tumors initiate an angiogenic switch, the molecular basis of this process remains incompletely understood. Here we show that the miRNA miR-132 acts as an angiogenic switch by targeting p120RasGAP in the endothelium and thereby inducing neovascularization. We identified miR-132 as a highly upregulated miRNA in a human embryonic stem cell model of vasculogenesis and found that miR-132 was highly expressed in the endothelium of human tumors and hemangiomas but was undetectable in normal endothelium. Ectopic expression of miR-132 in endothelial cells in vitro increased their proliferation and tube-forming capacity, whereas intraocular injection of an antagomir targeting miR-132, anti–miR-132, reduced postnatal retinal vascular development in mice. Among the top-ranking predicted targets of miR-132 was p120RasGAP, which we found to be expressed in normal but not tumor endothelium. Endothelial expression of miR-132 suppressed p120RasGAP expression and increased Ras activity, whereas a miRNA-resistant version of p120RasGAP reversed the vascular response induced by miR-132. Notably, administration of anti–miR-132 inhibited angiogenesis in wild-type mice but not in mice with an inducible deletion of Rasa1 (encoding p120RasGAP). Finally, vessel-targeted nanoparticle delivery1 of anti–miR-132 restored p120RasGAP expression in the tumor endothelium, suppressed angiogenesis and decreased tumor burden in an orthotopic xenograft mouse model of human breast carcinoma. We conclude that miR-132 acts as an angiogenic switch by suppressing endothelial p120RasGAP expression, leading to Ras activation and the induction of neovascularization, whereas the application of anti–miR-132 inhibits neovascularization by maintaining vessels in the resting state.

In the developing myocardium, vascular endothelial growth factor (VEGF)–dependent neovascularization occurs by division of existing vessels, a process that persists for several weeks following birth. During this remodeling phase, mRNA expression of β3 integrin in the heart decreases significantly as vessel maturation progresses. However, in male mice lacking β3, coronary capillaries fail to mature and continue to exhibit irregular endothelial thickness, endothelial protrusions into the lumen, and expanded cytoplasmic vacuoles. Surprisingly, this phenotype was not seen in female β3-null mice. Enhanced VEGF signaling contributes to the β3-null phenotype, because these vessels can be normalized by inhibitors of VEGF or Flk-1. Moreover, intravenous injection of VEGF induces a similar angiogenic phenotype in hearts of adult wild-type mice. These findings show a clear vascular phenotype in the hearts of mice lacking β3 and suggest this integrin plays a critical role in coronary vascular development and the vascular response to VEGF.

Liver fibrosis is characterized by an increased deposition of extracellular matrix proteins, including collagen type I, by activated hepatic stellate cells (HSCs). Previous studies have shown that this increase is mediated primarily by a post-transcriptional mechanism. In particular, the RNA-binding protein alphaCP binds to the alpha1(I) collagen 3'-untranslated region (UTR) and stabilizes this RNA in activated, but not quiescent, HSCs. This study examines the role of alphaCP in the decay of transcripts containing the collagen 3'-UTR in extracts obtained from NIH fibroblasts and quiescent and activated HSCs. Using an in vitro decay system, alphaCP binding activity was competed out with the addition of wild type oligonucleotides, but not with mutant oligonucleotides. Competition of alphaCP binding activity increased the rate of decay of wild type transcripts containing the alphaCP 3'-UTR binding site, but not of transcripts containing a mutated binding site. Quiescent HSC extracts contain no alphaCP binding activity and have no difference in the rate of decay of transcripts with wild type and mutant binding sites for alphaCP. The addition of recombinant alphaCP was sufficient to increase the half-life of the wild type transcript, whereas that of the mutant transcript was minimally changed. In vitro decay assays performed with activated HSC extracts that contain alphaCP binding activity demonstrate a markedly reduced decay rate of wild type compared with mutant transcripts. In vivo small interfering RNA experiments targeting alphaCP showed a reduction of the binding activity of alphaCP and a concomitant reduction in intracellular levels of alpha1(I) collagen messenger RNA. In conclusion, this study demonstrates the direct role of alphaCP in the stabilization of alpha1(I) collagen messenger RNA by blocking RNA degradation in activated HSCs.

Purpose: To investigate the effects of talazoparib alone and in combination with temozolomide (TMZ) on prostate cancer cells Introduction: Talazoparib is a potent, orally bioavailable, small molecule inhibitor of PARP (poly-ADP ribose polymerase) enzyme activity that also traps PARP protein onto DNA at sites of DNA damage; both activities result in cancer cell death (Murai et al. Mol Cancer Ther. 2014;13:433-43). Talazoparib is currently under investigation for clinical activity in patients who have locally advanced and/or metastatic breast cancer with germline BRCA mutations (EMBRACA; Clinical Trials.gov; NCT01945775). In the current nonclinical study, talazoparib demonstrated potent antitumor effects on androgen receptor (AR)-positive prostate cancer cells expressing both full length AR (LNCaP cells) as well as the androgen-independent splice variant, AR-V7 (22RV1 cells), when used either as a single agent or in combination with low, noncytotoxic concentrations of TMZ, a DNA alkylating therapeutic. Methods: Viability of prostate cancer cells (LNCaP and 22RV1 cells) after 7 days of treatment was measured by Cell Titer Glo (Promega) after treatment with vehicle, talazoparib, TMZ or a combination of the two. DNA damage was determined by immunofluorescence against gH2AX (EMD Millipore), and the amount of PARP trapped on DNA, determined by Western Blot analysis of chromatin probed with antibodies specific for PARP. Cellular homeostasis was determined by flow cytometry analysis for cell cycle stage, and apoptosis indicated by Annexin V staining (BD Pharmingen) or caspase activation with Caspase Glo (Promega) assays. Nucleotide adducts, N7 methyl-2'-deoxyguanosine (N7-MedG) and O6-Methyl-2'-deoxyguanosine (O6-MedG), from 1-, 2- and 4-hour TMZ-treated calf thymus DNA and genomic DNA from LNCaP cells treated with TMZ were detected after DNA hydrolysis and quantitated by LC-mass spectrometry in multiple reaction monitoring mode. Results: Talazoparib treatment resulted in a concentration-dependent inhibition of cell viability in LNCaP (AR wildtype) and 22RV1 (AR-V7) cells. Talazoparib's antitumor effects were greatly enhanced when combined with noncytotoxic TMZ concentrations. In a cell-free study using calf thymus DNA, TMZ generated both N7-MedG and O6-MedG DNA adducts, with the N7-MedG being more abundant than the O6-MedG. Analysis of DNA from LNCaP cells treated with TMZ generated detectable N7-MedG DNA adducts. Low, noncytotoxic concentrations of TMZ potentiated the effects of talazoparib on prostate cancer cell viability independent of AR variant status. Treatment with talazoparib alone and in combination with TMZ was associated with increased gH2AX staining (DNA damage) and enhanced PARP-DNA trapping complexes. The combination treatment also induced an increase in apoptotic cells at early time points. Conclusion: Talazoparib is cytotoxic to prostate cancer cells as a single agent in nonclinical cell based models. Herein, we demonstrate that combining noncytotoxic doses of TMZ with talazoparib improves efficacy in killing of LNCaP and 22RV1 cells. The increased efficacy corresponds with increased DNA-damage and increased trapped PARP-DNA complexes. Low, noncytotoxic doses of TMZ are shown to generate DNA damage sites in vitro and in treated LNCaP cells, providing a mechanistic basis for the combination effect of talazoparib and TMZ. Together, these nonclinical data provide scientific rationale for a clinical trial strategy of combining talazoparib with low, noncytotoxic doses of TMZ to enhance efficacy in prostate cancer. Citation Format: Jeffrey N. Lindquist, Vernon T. Phan, Eduardo Riquelme, Kakoli Mukherjee, Olivia Farías, Ashu Gupta, Mohd Raja, Roopa Rai, Hirdesh Uppal, Andrew A. Protter. Talazoparib efficacy is enhanced by noncytotoxic doses of temozolomide-mediated DNA damage in prostate cancer cell lines [abstract]. In: Proceedings of the AACR Special Conference on DNA Repair: Tumor Development and Therapeutic Response; 2016 Nov 2-5; Montreal, QC, Canada. Philadelphia (PA): AACR; Mol Cancer Res 2017;15(4_Suppl):Abstract nr B30.

The formation of the multicellular vascular system is critical to the growth, development, and viability of an organism, and many embryonic lethal mouse knockouts are due to vascular defects. Unfortunately, the complex nature, and many cell types involved in vasculogenesis and angiogenesis has stymied in vitro models of vascular formation. This unit describes a system that allows human embryonic stem cells to differentiate and spontaneously form vascular networks via both vasculogenesis and angiogenesis in the context of the three germ layers. Curr. Protoc. Stem Cell Biol. 12:1F.9.1-1F.9.6. © 2010 by John Wiley & Sons, Inc. Keywords: endothelial cells; vascular patterning; differentiation; angiogenesis; embryoid body

We have examined the kinetics of processing of the HIV-1 Gag-Pro-Pol precursor in an in vitro assay with mature protease added in trans. The processing sites were cleaved at different rates to produce distinct intermediates. The initial cleavage occurred at the p2/NC site. Intermediate cleavages occurred at similar rates at the MA/CA and RT/IN sites, and to a lesser extent at sites upstream of RT. Late cleavages occurred at the sites flanking the protease (PR) domain, suggesting sequestering of these sites. We observed paired intermediates indicative of half- cleavage of RT/RH site, suggesting that the RT domain in Gag-Pro-Pol was in a dimeric form under these assay conditions. These results clarify our understanding of the processing kinetics of the Gag-Pro-Pol precursor and suggest regulated cleavage. Our results further suggest that early dimerization of the PR and RT domains may serve as a regulatory element to influence the kinetics of processing within the Pol domain.

Angiotensin II (Ang II) is a pro-oxidant and fibrogenic cytokine. We investigated the role of NADPH oxidase in Ang II-induced effects in hepatic stellate cells (HSCs), a fibrogenic cell type. Human HSCs express mRNAs of key components of nonphagocytic NADPH oxidase. Ang II phosphorylated p47phox, a regulatory subunit of NADPH oxidase, and induced reactive oxygen species formation via NADPH oxidase activity. Ang II phosphorylated AKT and MAPKs and increased AP-1 DNA binding in a redox-sensitive manner. Ang II stimulated DNA synthesis, cell migration, procollagen alpha1(I) mRNA expression, and secretion of TGF-beta1 and inflammatory cytokines. These effects were attenuated by N-acetylcysteine and diphenylene iodonium, an NADPH oxidase inhibitor. Moreover, Ang II induced upregulation of genes potentially involved in hepatic wound-healing response in a redox-sensitive manner, as assessed by microarray analysis. HSCs isolated from p47phox-/- mice displayed a blunted response to Ang II compared with WT cells. We also assessed the role of NADPH oxidase in experimental liver fibrosis. After bile duct ligation, p47phox-/- mice showed attenuated liver injury and fibrosis compared with WT counterparts. Moreover, expression of smooth muscle alpha-actin and expression of TGF-beta1 were reduced in p47phox-/- mice. Thus, NADPH oxidase mediates the actions of Ang II on HSCs and plays a critical role in liver fibrogenesis.

Hepatic stellate cells are the major source of extracellular matrix proteins in hepatic fibrosis, including Type I collagen. In response to liver injury, the hepatic stellate cells change from a quiescent to an activated phenotype. This activation process includes a phenotypic change to a myofibroblast-like cell, increased proliferation rate, loss of retinoid stores, increased production of extracellular matrix proteins, chemokines, and cytokines, and contractility. Ongoing studies are characterizing the genes that are differentially expressed in the quiescent and activated hepatic stellate cells. We have also investigated the regulation of Type I collagen expression, the cleavage of collagen propeptides, and the formation of collagen cross-links. Understanding these pathways may provide new insights into the molecular pathogenesis of hepatic fibrosis.

The stability of collagen alpha1(I) mRNA is regulated by its 5' stem-loop, which binds a cytoplasmic protein in a cap-dependent manner, and its 3'-untranslated region (UTR), which binds alphaCP. When cultured in a three-dimensional gel composed of type I collagen, mouse fibroblasts had decreased collagen alpha1(I) mRNA steady-state levels, which resulted from a decreased mRNA half-life. In cells cultured in gel, hybrid mouse-human collagen alpha1(I) mRNA with a wild-type 5' stem-loop decayed faster than the same mRNA with a mutated stem-loop. When the 5' stem-loop was placed in a heterologous mRNA, the mRNA accumulated to a lower level in cells grown in gel than in cells grown on plastic. This suggests that the 5' stem-loop down-regulates collagen alpha1(I) mRNA. Protein binding to the 5' stem-loop was reduced in cells grown in gel, which was associated with destabilization of the collagen alpha1(I) mRNA. In addition to the binding of a cytoplasmic protein, there was also a nuclear binding activity directed to the collagen alpha1(I) 5' stem-loop. The nuclear binding was increased in cells grown in gel, suggesting that it may negatively regulate expression of collagen alpha1(I) mRNA. Binding of alphaCP, a protein involved in stabilization of collagen alpha1(I) mRNA, was unchanged by the culture conditions.