Rationale and Goal: Endothelial cells (ECs) are quiescent and critical for maintaining homeostatic functions of the mature vascular system, while disruption of quiescence is at the heart of endothelial to mesenchymal transition (EndMT) and tumor angiogenesis. Here, we addressed the hypothesis that KLF4 maintains the EC quiescence.Methods and Results: In ECs, KLF4 bound to KLF2, and the KLF4-transctivation domain (TAD) interacted directly with KLF2. KLF4-depletion increased KLF2 expression, accompanied by phosphorylation of SMAD3, increased expression of alpha-smooth muscle actin (αSMA), VCAM-1, TGF-β1, and ACE2, but decreased VE-cadherin expression. In the absence of Klf4, Klf2 bound to the Klf2-promoter/enhancer region and autoregulated its own expression. Loss of EC-Klf4 in RosamT/mG::Klf4fl/fl::Cdh5CreERT2 engineered mice, increased Klf2 levels and these cells underwent EndMT. Importantly, these mice harboring EndMT was also accompanied by lung inflammation, disruption of lung alveolar architecture, and pulmonary fibrosis.Conclusion: In quiescent ECs, KLF2 and KLF4 partnered to regulate a combinatorial mechanism. The loss of KLF4 disrupted this combinatorial mechanism, thereby upregulating KLF2 as an adaptive response. However, increased KLF2 expression overdrives for the loss of KLF4, giving rise to an EndMT phenotype.

The nitric oxide synthases (NOS; EC 1.14.13.39) use L-arginine as a substrate to produce nitric oxide (NO) as a by-product in the tissue microenvironment. NOS1 represents the predominant NO-producing enzyme highly enriched in the brain and known to mediate multiple functions, ranging from learning and memory development to maintaining synaptic plasticity and neuronal development, Alzheimer’s disease (AD), psychiatric disorders and behavioral deficits. However, accumulating evidence indicate both canonical and non-canonical roles of NOS1-derived NO in several other tissues and chronic diseases. A better understanding of NOS1-derived NO signaling, and identification and characterization of NO-metabolites in non-neuronal tissues could become useful in diagnosis and prognosis of diseases associated with NOS1 expression. Continued investigation on the roles of NOS1, therefore, will synthesize new knowledge and aid in the discovery of small molecules which could be used to titrate the activities of NOS1-derived NO signaling and NO-metabolites. Here, we address the significance of NOS1 and its byproduct NO in modifying pathophysiological events, which could be beneficial in understanding both the disease mechanisms and therapeutics.

RationaleThe human epigenome is plastic. The goal of this study was to address if fibroblast cells can be epigenetically modified to promote neovessel formation.Methods and resultsHere, we used highly abundant human adult dermal fibroblast cells (hADFCs) that were treated with the chromatin-modifying agents 5-aza-2'-deoxycytidine and trichostatin A, and subsequently subjected to differentiation by activating Wnt signaling. Our results show that these epigenetically modified hADFCs increasingly expressed β-catenin, pluripotency factor octamer-binding transcription factor-4 (OCT4, also known as POU5F1), and endothelial cell (EC) marker called vascular endothelial growth factor receptor-2 (VEGFR-2, also known as Fetal Liver Kinase-1). In microscopic analysis, β-catenin localized to cell-cell contact points, while OCT4 was found to be localized primarily to the nucleus of these cells. Furthermore, in a chromatin immunoprecipitation experiment, OCT4 bound to the VEGFR-2/FLK1 promoter. Finally, these modified hADFCs also transduced Wnt signaling. Importantly, on a two-dimensional (2D) gel substrate, a subset of the converted cells formed vascular network-like structures in the presence of VEGF.ConclusionChromatin-modifying agents converted hADFCs to OCT4+ and VEGFR-2+ capillary tube-forming cells in a 2D matrix in VEGF-dependent manner.

The ability of genome editing tool to correct genetic disease, or edit the tumor causing gene, will not only benefit human race, but also plants and animal kingdom. The CRISPR/Cas9 system can be used to target specific genomic loci by single guide RNAs (sgRNAs). Using publicly available literature, we have modeled the structure of Streptococcus pyogenes Cas9 molecule in complex with sgRNA and its target DNA at 2.5 A° resolution. The crystal structure determination revealed a two-lobed architecture made of: (a) target recognition and (b) nuclease lobes, positioning the sgRNA:DNA hetero-duplex in a positively charged groove at their interface. While the recognition lobe interacts with the sgRNA and DNA, the nuclease lobe contains the HNH and RuvC nuclease domains that are responsible for cleavage of the complementary and non-complementary target DNA strands, respectively. The nuclease lobe also harbors a carboxyl-terminal domain to provide additional specificity for interaction with the protospacer adjacent motif (PAM) DNA sequence. This structural analyses show the molecular mechanism of RNA-guided DNA targeting by Cas9, thus providing the CRIPSR/Cas9 genetic machinery its ability to edit specific DNA sequence. (Key Ref: Nishimasu H et al., Cell. 2014;156:935-49) Citation Format: Anita Wary. Modeling the CRISPR/Cas9 structural complex with sgRNA and DNA [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1374.

The vascular endothelial growth factor receptor-2 (VEGFR2) is a receptor tyrosine kinase that is expressed highly in endothelial cells (ECs). By binding to VEGF165 (VEGF-A) VEGFR2 signals in ECs mediate the formation of new blood vessels, a process known as angiogenesis. Growth of tumor and metastasis depends on angiogenic activities of ECs. Thus, an agent that blocks VEGFR2 signaling specifically in tumor ECs could inhibit tumor growth and metastasis. The IMC-1121B is an antiangiogenic agent that inhibits the VEGFR2 signaling. Here we modeled the structure of the 1121B Fab fragment in complex with domain-3 of VEGFR2, and the structure of a different neutralizing anti-VEGFR2 antibody, 6.64, also in complex with VEGFR2 domain-3. The two Fab fragments interact at opposite ends of VEGFR2 domain-3; 1121B blocks VEGF binding, whereas 6.64 appears to prevent receptor dimerization by perturbing the domain-3:domain-4 interface. Blocking of VEGFR2 signaling activities by two different antibodies could be a powerful approach to block angiogenesis, thereby inhibiting growth of tumor and metastasis. Key reference: Franklin MC et al., Structure 2011;19:1097-1107. Citation Format: Anita Wary. Structural analysis of VEGFR2 function blocking antibodies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2046.