Identification of a physiologic vasculogenic fibroblast state to achieve tissue repair.

Tissue injury to skin diminishes miR-200b in dermal fibroblasts. Fibroblasts are widely reported to directly reprogram into endothelial-like cells and we hypothesized that miR-200b inhibition may cause such changes. We transfected human dermal fibroblasts with anti-miR-200b oligonucleotide, then using single cell RNA sequencing, identified emergence of a vasculogenic subset with a distinct fibroblast transcriptome and demonstrated blood vessel forming function in vivo. Anti-miR-200b delivery to murine injury sites likewise enhanced tissue perfusion, wound closure, and vasculogenic fibroblast contribution to perfused vessels in a FLI1 dependent manner. Vasculogenic fibroblast subset emergence was blunted in delayed healing wounds of diabetic animals but, topical tissue nanotransfection of a single anti-miR-200b oligonucleotide was sufficient to restore FLI1 expression, vasculogenic fibroblast emergence, tissue perfusion, and wound healing. Augmenting a physiologic tissue injury adaptive response mechanism that produces a vasculogenic fibroblast state change opens new avenues for therapeutic tissue vascularization of ischemic wounds.

Inducible miR-1224 silences cerebrovascular Serpine1 and restores blood flow to the stroke-affected site of the brain.

The α-tocotrienol (TCT) form of natural vitamin E is more potent than the better known α-tocopherol against stroke. Angiographic studies of canine stroke have revealed beneficial cerebrovascular effects of TCT. This work seeks to understand the molecular basis of such effect. In mice, TCT supplementation improved perfusion at the stroke-affected site by inducing miR-1224. miRNA profiling of a laser-capture-microdissected stroke-affected brain site identified miR-1224 as the only vascular miR induced. Lentiviral knockdown of miR-1224 significantly blunted the otherwise beneficial effects of TCT on stroke outcomes. Studies on primary brain microvascular endothelial cells revealed direct angiogenic properties of miR-1224. In mice not treated with TCT, advance stereotaxic delivery of an miR-1224 mimic to the stroke site markedly improved stroke outcomes. Mechanistic studies identified Serpine1 as a target of miR-1224. Downregulation of Serpine1 augmented the angiogenic response of the miR-1224 mimic in the brain endothelial cells. The inhibition of Serpine1, by dietary TCT and pharmacologically, increased cerebrovascular blood flow at the stroke-affected site and protected against stroke. This work assigns Serpine1, otherwise known to be of critical significance in stroke, a cerebrovascular function that worsens stroke outcomes. miR-1224-dependent inhibition of Serpine1 can be achieved by dietary TCT as well as by the small-molecule inhibitor TM5441.

Driving adult tissue repair via re-engagement of a pathway required for fetal healing.

Fetal cutaneous wound closure and repair differ from that in adulthood. In this work, we identify an oxidant stress sensor protein, nonselenocysteine-containing phospholipid hydroperoxide glutathione peroxidase (NPGPx), that is abundantly expressed in normal fetal epidermis (and required for fetal wound closure), though not in adult epidermis, but is variably re-induced upon adult tissue wounding. NPGPx is a direct target of the miR-29 family. Following injury, abundance of miR-29 is lowered, permitting a prompt increase in NPGPx transcripts and protein expression in adult wound-edge tissue. NPGPx expression was required to mediate increased keratinocyte migration induced by miR-29 inhibition in vitro and in vivo. Increased NPGPx expression induced increased SOX2 expression and ß-catenin nuclear localization in keratinocytes. Augmenting physiologic NPGPx expression via experimentally induced miR-29 suppression, using cutaneous tissue nanotransfection or targeted lipid nanoparticle delivery of anti-sense oligonucleotides, proved to be sufficient to overcome the deleterious effects of diabetes on this specific pathway to enhance tissue repair.

Neurogenic tissue nanotransfection in the management of cutaneous diabetic polyneuropathy.

This work rests on our recent report on the successful use of tissue nanotransfection (TNT) delivery of Ascl1, Brn2, and Myt1l (TNTABM) to directly convert skin fibroblasts into electrophysiologically active induced neuronal cells (iN) in vivo. Here we report that in addition to successful neurogenic conversion of cells, TNTABM caused neurotrophic enrichment of the skin stroma. Thus, we asked whether such neurotrophic milieu of the skin can be leveraged to rescue pre-existing nerve fibers under chronic diabetic conditions. Topical cutaneous TNTABM caused elevation of endogenous NGF and other co-regulated neurotrophic factors such as Nt3. TNTABM spared loss of cutaneous PGP9.5+ mature nerve fibers in db/db diabetic mice. This is the first study demonstrating that under conditions of in vivo reprogramming, changes in the tissue microenvironment can be leveraged for therapeutic purposes such as the rescue of pre-existing nerve fibers from its predictable path of loss under conditions of diabetes.

A high energy phosphate jump - From pyrophospho-inositol to pyrophospho-serine.

Inositol pyrophosphates (PP-IPs) are a class of energy rich metabolites present in all eukaryotic cells. The hydroxyl groups on these water soluble derivatives of inositol are substituted with diphosphate and monophosphate moieties. Since the discovery of PP-IPs in the early 1990s, enormous progress has been made in uncovering pleiotropic roles for these small molecules in cellular physiology. PP-IPs exert their effect on proteins in two ways - allosteric regulation by direct binding, or post-translational regulation by serine pyrophosphorylation, a modification unique to PP-IPs. Serine pyrophosphorylation is achieved by Mg2+-dependent, but enzyme independent transfer of a ß-phosphate from a PP-IP to a pre-phosphorylated serine residue located in an acidic motif, within an intrinsically disordered protein sequence. This distinctive post-translational modification has been shown to regulate diverse cellular processes, including rRNA synthesis, glycolysis, and vesicle transport. However, our understanding of the molecular details of this phosphotransfer from pyrophospho-inositol to generate pyrophospho-serine, is still nascent. This review discusses our current knowledge of protein pyrophosphorylation, and recent advances in understanding the mechanism of this important yet overlooked post-translational modification.

Pharmacophore based 3D-QSAR modeling, virtual screening and docking for identification of potential inhibitors of ß-secretase.

The enzyme ß-secretase-1 is responsible for the cleavage of the amyloid precursor protein, a vital step in the process of the formation of amyloid-ß peptides which are known to lead to neurodegeneration causing Alzheimer's disease. Challenges associated with toxicity and blood brain permeation inability of potential inhibitors, continue to evade a successful therapy, thus demanding the search and development of highly active and effective inhibitors. Towards these efforts, we used a ligand based pharmacophore model generation from a dataset of known inhibitors whose activities against ß-secretase hovered in the nano molar range. The identified 5 feature pharmacophore model, AHHPR, was validated via three dimensional quantitative structure activity relationship as indicated by r2, q2 and Pearson R values of 0.9013, 0.7726 and 0.9041 respectively. For a dataset of compounds with nano molar activity, the important pharmacophore features present in the current model appear to be similar with those observed in the models resulting from much wider activity range of inhibitors. Virtual screening of the ChemBridge CNS-Set™, a database having compounds with a better suitability for central nervous system based disorders followed by docking and analysis of the ligand protein interactions resulted in the identification of eight prospective compounds with considerable diversity. The current pharmacophore model can thus be useful for the identification, design and development of potent ß-secretase inhibitors which by optimization can be potential therapeutics for Alzheimer's disease.

Identification of abelson tyrosine kinase inhibitors as potential therapeutics for Alzheimer's disease using multiple e-pharmacophore modeling and molecular dynamics.

Efforts to combat Alzheimer's disease are focused predominantly on inhibiting the activity of the enzyme(s) that have been identified to be responsible for the production of the amyloid-forming peptide. However, the inherent complexity associated with the network of pathways leading to the disease may involve additional targets for designing effective therapies. Recent experimental findings have identified abelson tyrosine kinase, a non-receptor kinase as a new target for Alzheimer's. In this work, we employed energy optimized multiple pharmacophore modeling strategy from multiple c-Abl structures bound with ligands in the inactive ATP binding conformation (DFG-out). Virtual screening followed by docking of molecules from ChemBridge resulted in the identification of 10 best scoring molecules. MD simulations of the top three complexes revealed that Compound A, C are the most stable complexes with the most persistent protein-ligand interactions consistent with the calculated binding affinities for the top three compounds. Given the implied role of c-Abl not only in AD but in Parkinson's disease, the identified compounds may serve as leads for effective neurotherapeutics.

Multiple e-Pharmacophore Modeling Combined with High-Throughput Virtual Screening and Docking to Identify Potential Inhibitors of ß-Secretase(BACE1).

ß-Secretase (BACE1) is an aspartate protease involved in the production of amyloid-ß a major peptide responsible for the pathogenesis of Alzheimer's disease. Given its role in the formation of amyloids leading to Alzheimer's disease, it has been a major therapeutic target for intervention and has been a challenge in the past and the progress has been very slow. More than hundred crystal structures with inhibitors are available in the protein data bank. Many strategies for drug design have been employed in the design of numerous diverse ligands for this target and many have failed due to undesirable drug properties primarily the inability to cross the blood-brain barrier. In the present work we attempted to consider multiple crystal structures with bound inhibitors showing affinity in the range of 2-210 nM efficacy and optimize the pharmacophoric requirement based on the energy involved in binding termed as e-pharmacophore mapping. A high throughput screening combined with molecular docking, ADMET predictions, logP values and in vitro assay led to the identification of 7 potential compounds showing inhibition at 10µM which could be further developed as novel inhibitors for ß-secretase.