Lian Guo, PhD: Okay, make sure, yeah, it works. So you can see it's on the screen about me. I'm in Department of Surgery, but I'm not a surgeon; I'm PhD, and I'm also appointed in the Department of Physiology and Cell Biology. So I have two hats. Also, something strange about my research: I have a research project in two distinct research fields, vascular biology and retinal neuroscience. [Text on screen Snapshots of the Guo Lab Research Lian Guo PhD Associate Professor Depts of Surgery and Physiology/Cell Biology] So first, I want to introduce my vascular project. [Text on screen Vascular Biology] So we target the disease called restenosis; this is a recurrent disease that occurs after the surgical procedures to treat cardiovascular disease. So, to treat vascular disease, at the same time, that injury cause the formation of new intima lesions that blocks blood flow, so that's a restenosis. And the picture we're looking at is normal blood vessel artery, so smooth muscle cells, those are the blocking the building blocks in the blood vessel wall that provide strength and elasticity to the artery. [Text on screen Neointima/Restenosis after surgical injury] [Drawing of the Lumen with smooth muscle cells labeled] But after surgical injury, they change phenotypes and they become highly proliferative, forming this thick layer of neointima lesion, and over time, the neointima build-up and blocks the blood flow. So our interest is to identify novel mechanism of restenosis. [Image with the neointima labeled, the smooth muscle cells, and the re-narrowing of the neointima] So, we found that BET epigenetic readers, this family of proteins, represent a novel regulator of this disease. And BET proteins read or recognize modification of histone, so that's a chromatin modification, acetylation, and the BET proteins translate this chromatin marking to gene expression. [Image on screen Nucleosome with BET reader attached to Pol-II approaching a histone] And the nice thing is, so these family of proteins recently were identified to be a drug target, and a selective drug, JQ1, can block the activity of the BET proteins. [Image on screen Nucleosome with BET reader attached to Pol-II labeled JQ1 approaching a histone] So, we use the animal model to study the function of the BET family proteins in restenosis or in the formation of neointima lesions. So, this picture is a part of the blood vessel wall, uninjured. After injury, at three days after injury, you see those brown dots, that is the BET protein, BRD4. One of the BET proteins it's upper regulated. Then at seven days, it's dramatically upregulated, you see the brown color, that's in the neointima region, and also 14 days. So in this animal model, if we don't do treatment, so the injury cause the formation of a thick layer of a neointima. [Text on screen BET inhibitor JQ1 abrogates neointima] [Image of BRD4 Staining after uninjured, injured after 3 days, 7 days, and 14 days] And if we treat the animals with JQ1, the inhibitor of BET proteins, there is little neointima formed. And in vitro, so in cell dish, culture dish, these inhibitor of BET proteins also inhibit smooth muscle cell proliferation. [Image on screen neointima after vehicle and JQ1 (500 microgram/rat)] [Graph comparing the relationship between concentration of JQ1 and proliferation] So, currently, we are looking for the transcription factors that associate with BET proteins to study the mechanism, their regulatory mechanism, and by CHIPseq, that is, chromatin immunoprecipitation and bioinformatics analysis, we found STAT3 is top-scored. [Text on screen Genome-Wide ChIPseq analysis] [Image on screen A- Graph displaying co-immunoprecipitation of BRD4 and STAT3 B- STAT3 with P-value of le-58 with letters for DNA code are above those of Elk1 with a P-value of le-50] And we also have a preliminary data showing stat three co-immunoprecipitates with BRD4. So, the potential PhD projects will be to identify BET-associated transcription factors. [Text on screen Co-immunoprecipitation of BRD4 and STAT3] [Image on screen Western blot results are displayed comparing IP: IgG and IP:BET4 (BRD4) STAT3 with image of western blot results TNF alpha - + + Jq1 - - +.] So we have a STAT3 as a candidate, and we think probably there are more. And we also want to explore BET functions in other vascular diseases because it's very little studied in the vascular field. For example, aneurysm, that's another disease. [Text on screen PhD projects: -Identify BET-associated transcription factors -Explore BET functions in other vascular diseases] Now, retinal project. [Text on screen Retinal Neuroscience] So we focus on a protein called sigma-1 receptor. So this protein is a drug target for hallucinogen and cocaine, and this protein has been found to be protective for retinal neurons. [Text on screen The sigma-1 receptor: -A drug target for retinal neuroprotection] So, to identify the precise subcellular localization, we improved a method called APEX2-enhanced electron microscopy. So, the way we did it, we fuse or link GFP protein and APEX2 to the sigma-1 receptor. And APEX-2, which is enzyme that is like a horseradish peroxidase, that catalyzes free-radical reaction with DAB, this chemical, and polymerizes together with the gold/silver precipitation. [Text on screen APEX2-enhanced EM] [Graph displaying the APEX2-enhanced gold/silver particle EM] We can see some dark dots, dark particles. So here you see the dark dots represent where sigma-1 receptor is located, and we see sigma-1 receptor is predominantly localized on the Network, you see those structures. [Image on screen: Black and white photo of Sigma-1R (dark dots) with ER labeled in yellow.] And we found sigma-1 receptor also localized on the nuclear envelope. So it's a really high resolution, so this method greatly enhance the clarity of the symbol receptor localization. And, we also found sigma receptor on the nucleoplasmic reticulum, so this is our novel funding. [Image on screen: Zoomed in black and white image of the nucleoplasmic reticulum with Nu, NE, NR, and ER labeled and two yellow arrows.] And taking a closer look, you can see the sigma-1 receptor in ER, also localized in ER that juxtaposed to lysosomes. So, lysosome is an organelle that's very important for autophagy. Autophagy is a cellular process that for cellular waste recycling. [Image on screen: Zoomed in black and white image with Nucleus and 2 Lyso's labeled with a yellow arrow poiting at a small black dot cluster.] Last year, the Nobel Prize was given to Japanese scientists who first studied autophagy, and here is schematic for autophagy. So basically, autophagosome that's an organelle that wraps up the cellular waste and fused with the lysosomes, the green bubble, so the cellular waste, like proteins and damaged mitochondria, got degraded and recycled. [Image showing bubbles inside a cell, labeled ER, Lyso, and Autophagos] And we know that sigma-1 receptor is Membrane. And we hypothesized that a sigma-1 receptor is important for the organelle fusion, the autophagosome-lysosome, for this fusion, of course, based on some evidence. To study this, we made a sigma-1 receptor knockout cell line using this CRISPR-Cas9 technology. [Text on screen: KO by CRISPR-Cas9] [Image on screem: CRISPR-Cas9 successfully knocked out two proteins—PGRMC1 and Sigma-1R—since the dark protein bands seen in normal cells (WT) disappear in the knockout cells (KO) displayed via western blot.] And to visualize the fusion process, the fusion of the autophagosome and the lysosome, we use this construct, RFP-GFP tandem, to label autophagosome. [Text on screen GFP fluoresence quenching in lysosomes is impaired by Sigma1R KO.] [Image on screen: A wildtype cell with many small, scattered bright spots indicating normal lysosome activity where green light is quenched] The idea is that if autophagosomes can fuse with lysosome, then the acidic pH inside the lysosome will quench GFP, so the green color is gone, so only red is left. So that's why we see many red dots that indicates that in the wild type cells, the autophagosome-lysosome fusion is normal. But in the knockout, you see, we see a lot of yellow, so why we see yellow? Because that suggests that the autophagosome cannot fuse with lysosome, that means without the lysosome acidic pH, quenching GFP is there, so red and green both are there with the yellow. [Image on screen: Sig1R Ko cell that appears mostly bright yellow, showing that green light is not quenched properly, indicating impaired lysosome function (NSC34 cells).] To further study the possible interaction of sigma-1 receptor with the fusion machinery proteins, we use a method called fluorescence complementation. So basically, it's a split GFP, two halves, so one half of the sigma-1 receptor, the other half on the potential interacting protein. If the two proteins are interacting or close to each other, so the two halves will reunite, and the GFP fluorescence is restored. [Image on screen: A protein called Sig1R starting in endoplasmic reticulum (ER) and sends a signal to the lysosome, where another curly protein helps guide that signal into the lysosome] So if we see green, likely the two proteins are interacting, so we see green with this pair, sigma-1 receptor and VAMP8, VAMP8 is approaching in the organelle fuel machinery. [Image on screen: Sig1R in the endoplasmic reticulum connected by a green arrow and curly yellow structure to the lysosome] So, we also verified this result with a coimmunoprecipitation. [Text on screen: Possible Sigma1R-VAMP8 interaction.] [Imaging showing a scientific experiment suggesting that two proteins, Sigma1R and VAMP8, may physically interact in cells] I can't present all the data, but we also identify sigma-1 receptor who interact with other proteins in that fuel machinery. [Diagram describing the "docking" model] We got a model, it's a busy model, just simplify. So we think sigma-1 receptor serves as a docking site and brings the two organelles, autophagosome-lysosome, together, so facilitate their fusion. So, for this project, we have some fresh data showing that sigma-1 receptor is also important for lipid synthesis and storage. For example, in the knockout animal, we feed with a high-fat troll. In the knockout, the weight, body weight doesn't grow that fast, that potentially is a PhD project. And sigma-1 receptor has a brother, sigma-2 receptor; it's an enigmatic protein because it was, in the past 30 years, it was misidentified and misidentified again. But finally, so two months ago, this protein was cloned and identified as transmembrane protein 97, which is a very little-studied studied, only around 10 or 11 papers in PubMed. [Text on screen: PhD projects: -Sigma-1R function in lipid synthesis and storage -Sigma-2R (TMEM97) in retinal protection] So, we are making TMEM97 knockout mice. We want to study the sigma-2 receptor function in the retina, and our ultimate goal is to translate our research into the measures that can be used in the clinic. [Text on screen: Translational.] So we use a type of nanoparticle. You see, it's called a unimolecular nanoparticle because the whole thing is just a single molecule. And it has a core and has a hydrophobic core that can harbor hydrophobic drug and a hydrophilic shell that makes the nanoparticle highly water-soluble, and the cool thing is that you can attach anything to it, so it's a Christmas tree, you just have anything on it? Protein-ligand or fluorescent tag or biotin, it looks bulky here, but actually it's very small, it's tiny, just around 50 nanometer diameter, and it's a completely clear in solution. [Image on screen: Diagram of the unimolecualr micelle] So we load the drug to the nanoparticles and then suspend the nanoparticles in a hydrogel. [Image showing a drug molecule and electron microscope images of tiny round nanoparticles used in a nanoparticle/hydrogel drug delivery system] The hydrogel is a liquid at low temperature, but becomes gel at body temperature. So we use the animal model, use a balloon to injure the artery that will produce the neointima lesion. And we apply the drug in a nanoparticle and in the gel to outside of the artery, and three months later, so without treatment, you see, we have the artery has this thick layer of neointima lesion. [Image showing a close-up surgical view inside a rat, where shiny, pink tissue is visible along with a white tube and black sutures used to treat neointima in which the cartoid artery and balloon are labeled.] But with the drug treatment, with a nanoparticle, there is no new intima or very little left. So this system, the uniqueness about this system is that we can achieve the efficacy for three months, that's a rare feat. In the literature, people can rarely achieve that, the quantification. [Graph on screenshowing perivascular delivery effect on the neointima after 3 months -B Cttrl has a higher intima abrogate than resv -C Ctrl has a lower lumen area than resv D Ctril and Resv are relitively the same EEL length] [Image on screen -A control artery section after 3 months delivery -B resv artery cell after 3 months delivery] So that is the application of this drug delivery system for the vascular disease. Of course, we are not forgetting about the eye. So, using the same nanoparticle, but we modify differently, we attach a targeting protein as a green arch to a nanoparticle, red, so black dots are a drug. And so the targeting protein can accumulate nanoparticles to the ganglion neuron layer. Now, without targeting, they simply diffuse and get lost. [Image on screen: Targeted vs non-tarfeted delivery for protection of retinal neurons] Indeed, when we injected the system into a mouse eye, you see the white dots, so the nanoparticles are accumulated as a neuronal cell layer. Without the targeting, you can see much less. [Image on screen: Glowing retinal ganglion neurons along a curved eye layer labeled GCL, indicating targeted activity and non-targeted activity.] And look, let's see the protective effect for neurons, so each red dot that's a neuron cell, that's a ganglion neuron. And you see targeted nanoparticles, you have more cells, non-targeted, a lot smaller number. So here, quantification: white, that's a protected higher cell number; green, that's a lower number for this project. [Graph on screen: Number of RGCs based on day, comparing targeted and non-targeted over 14 days.] So, there is big challenge for the drug delivery to protect another important neuron in the retina, that's a photoreceptor, that's a light-sensitive neuron to make vision. And we want to further utilize these nanoparticles for protection of those neurons. [Text on screen Potential PhD project: Targeted nanoparticles for outer retinal neuron (photoreceptor) protection] So my collaborators: this is a real surgeon, Dr. Craig Kent, and Sara Gong, a biomedical engineer. [Text on screen Collaborators; -Dr. Craig Kent (Vascular Surgeon) -Dr. Sarah Gong (Biomedical Engineer) NIH funding OSU Startup] So my lab just relocated from University of Wisconsin, and I have a PhD student right now in the vascular biology project. I don't have a PhD student in the retina project, so I would like to invite your interest. And I have NIH grants and startup; don't worry about funding for next couple years, or next four or five years. Welcome. [Image on screen: Ohio State Logo in bottom left corner.]