Retinal Angiogenesis ANAT416_W2024 PDF

Basic and therapeutic aspects of retinal angiogenesis ANAT416_W2024 / March 26th 2024 Alexandre Dubrac Anatomy and Cell Biology Dept. / Human Genetics Dept. / McGill University [email protected] Associate professor Dépt. de Pathologie et Biologie Cellulaire / Dépt. d’ophtalmologie / Univers...

Basic and therapeutic aspects of retinal angiogenesis ANAT416_W2024 / March 26th 2024 Alexandre Dubrac Anatomy and Cell Biology Dept. / Human Genetics Dept. / McGill University [email protected] Associate professor Dépt. de Pathologie et Biologie Cellulaire / Dépt. d’ophtalmologie / Université de Montréal [email protected] Neurovascular patterning Lab Fetomaternal and Neonatal Pathologies Axis Sainte-Justine Hospital Neurovascular patterning: vascularization of the CNS RETINA BRAIN Understanding mechanisms governing angiogenesis and vascular morphogenesis in development, and diseases (Retinopathies and Stroke, and malformation). The anatomy of the vascular system Systemic circulation Pulmonary circulation Artery =They transport oxygenated blood to the tissues and organs. Vein=They transport deoxygenated blood to the right heart. Artery = They transport deoxygenated blood to the lungs (alveoli). Vein = They transport oxygenated blood to the left heart. Between artery and vein= Capillaries Site of gas exchange. where blood and tissues connect together Angiogenesis Formation of new blood vessels from existing vessels. Physiological or pathological phenomenon. ≠ Vasculogenesis: creation of “pipe” Potente et al, nature reviews, 2017 During early embryonic development, the formation of blood vessels de novo through the anastomosis of vascular rudiments (condensation of mesodermal cells that locally differentiate into endothelial and blood cells). intima - composed by endothelial cells, which are directly in contact with the bloodflow (they directly regualte information between blood and tissue) Angiogenesis and vascular heterogeneity BRAIN RETINA Formation of new blood vessels from existing vessels. Physiological or pathological phenomenon. LIVER BONES different blood vessels have different shapes in different organs ie. in brain, blood-brain barrier protects the neruons from the blood, only exists in CNS Enables the development of a specialized vasculature to meet the specific needs of each organ and facilitate its proper functioning. Augustin et al, science 2017 ex. in liver, you need fenestrated membranes (holes) to allow big molecules to enter the liver Angiogenesis Excessive Angiogenesis Adapted from Bae B. et al., Biomolecules 2021; D’Andrea L.D. et al., Dalton Trans. 2010 Angiogenesis and vascular remodeling the blood vessels have filipodia, to sense their surroundings and see where they must grow. Tip cells will guide the new vessel growth. The sprout will elongate, and you will get anastomosis = fusion of two tip cells to create an enlarged vessel diameter. Mouse Retina model has many advantages: smaller, retina is easy to access, shape of blood vessels is good for studying Retina vascularization aka Primitive vessels This stage in mice will start at birth Retina vascularization ALL OF THESE STEPS ARE DRIVEN BY HYPOXIA THis is a ball of neurons, that must be sliced and peeled open like a flower in order to be studied growth factors will be released, so there is a second step there is a formation of a deeper layer of blood vessels Neuronal layer Optic nerve Blood vessel the blood vessels sprout, and will start extending over the course of 7 days and there will be hypoxia on the surface of the retina not well known step, but there is an interaction between the vascularization across the whole retina Retina vascularization Vascular Remodelling, where primarly plexus extends to create arteries and veins in the retina Developing a transgenic mouse, to study process. Every day after brith, you can see this blood vessel network expanding and growing. By staining and imaging the capillaries. Simple and quantifiable analysis of mouse mutants or disease models You can see various phenotypes through this staining technique. Hypoxia-induced angiogenesis Capillaries extend along a VEGF gradient expressed by neural cells P1 VEGF-A Optic nerve Hypoxia extends the blood vessel network across the retinal tissue. Hypoxia Hypoxia-induced angiogenesis Capillaries extend along a VEGF gradient expressed by neural cells P5 VEGF-A Hypoxia Angiogenic front - contains the tip cells that guide and sprout the new information Arteries have less branching are are smaller than veins, Hypoxia-induced angiogenesis (hypoxia-inducible transcription factors) The level of VEGF-A would stay high if there was still hypoxia. Hypoxia inducing factor 1 (HIF-1) drives tumour growth, metastasis, and vascularizatoin Physician-scientist Gregg Semenza received the 2019 Nobel Prize in Physiology or Medicine P5 VEGF-A Hypoxia This work was reported in papers published in 1995 in the Proceedings of the National Academy of Sciences journal. It was submitted to and returned without review by the editors of Cell, Science, and Nature. This paper has now been cited over 6,000 times. Oxygen-induced HIF1⍺ degradation PVHL interaction iwth OH (under normoxia) will induce degratation von Hippel-Lindau tumor suppressor Hypoxia-induced HIF1⍺ stabilization THe pVHL cannot be recruited uner hypoxia, thus the protien will be stabilized, to lead to ativation instead of degradation HIF1⍺ triggers angiogenic transcriptomic reprogramming the first response under hypoxia is to create new vessels to supply oxygen as a survival instict: VEGF-R IS SUPER IMPORTANT Hypoxia-induced angiogenesis Capillaries extend along a VEGF gradient expressed by neural cells P5 VEGF-A Hypoxia Vascular Endothelial Growth Factor A (VEGF-A) If remove one alelle: If overexpressed: Vegf-a +/- : Embryonic lethal E9 Vegf-a KI : Embryonic lethal E12.5 How are tip cells selected? Both of these have an ebryonic lethaglity, vascularization, malformation VEGF signaling is crucial for vascular sprouting Tip cell/Stalk cell specification Notch isnt expressed in Tip cell, so if you activate Notch in Tip cells, you WON’T have differentiation and won’t have Tip cells Notch gets activated on the Stalk cells, which form the lumen (tip cells don’t) since we only want a few type cells, VEGFR2/3 is iexpressed - blocked by Notch, stopping them from becomming tip cells Metabolic heterogeneity regulates vascular sprouting they are glycolitic, for cytoskelatal remoddelling Pericytes This is to create the blood brain barrier 1. regruitment of peicytes, which help w sprouting and growht, help show they support tip Cell extension P5 retina SLIT2VEGF-A Hypoxia PC recruitment PDGFRβ PDGF-b PDGF-b perictye recruitmenet has a gradiaent in concentration as well = lowest Conc near breain, highest conc is closest to the retina Extra Cellular Matrix (ECM) Pericyte Angiogenesis Vessel stability Blood-Brain/Retina-Barrier Blood retina barrier VEGF-A: Identified as VPF - this will (vascular permeability factor) in 1983. happen at junction between two dcells and remodelling BRB is stablished at P10 this is what vascular leakage looks like: VEGF and Hypoxia can only induce sprouting on the superficial layers and not deeper sprouting Adapted from: Herbert and Stainier, Nat. Rev. Mol. Cell Bio., 2011 Venkatraman, L., Claesson-Welsh, L. Tumor Angiogenesis. 2019 Vascular leakage Chow B.W. & Gu C., Neuron 2017 Blood retina barrier Similar to Blood Brain Barrier (BBB) Paracellular CLDN5+ Transcellular MFSD2a+ Mazzoni J. et al, Neuron 2017 Biswas S. et al., Development 2020 Neuroretina vascularization mechanisms? P8-P14 Diving sprouts Tip Induced by VEGF Stalk WNT/NORRIN signaling high BRB? Tip VEGFA How these angiogenic sprouts can establish BRB? NORRIN Okabe K. et al, Cell, 2014 Ye X. et al, Cell, 2009 Chow B.W. & Gu C., Neuron 2017 Is it the same tip cell? Single-cell RNA sequencing (scRNAseq) Now that we have this, we can identify the heterogeneity of cells you put tags on individual cells, then you can have the information of genes for each individual geen Droplet-based single cell RNAseq tools: a practical guide. Salomon R. et al, Lab Chip Identification of temporal retinal EC heterogeneity P6-P10 WT retina a the types of cells will give us dots, and when we map it out, we will have cells that cluster together based on genetic similarities. Cones Mouse retina P6 P10 Immune cells Rods Vitreous chamber Microglia Retina Optic nerve MACS enrichment RPE Muller glia Pericytes Bipolar cells UMAP 2 Single cell RNA sequencing Taxonomy UMAP 1 d Zarkada G. et al. Dev Cell 2021 Amacrine cells ECs Astrocytes you can annotate and idenitfy different neurons Identification of temporal retinal EC heterogeneity P6-P10 ECs sub-clustering Cones Immune cells Rods Microglia RPE Muller glia Pericytes Amacrine cells UMAP 2 Bipolar cells UMAP 1 ECs Astrocytes D-Tip Cell S-Tip Cell Cell S-Tip Retina tip-cell heterogeneity 10μm 10μm 25μm D-Tip Cell Cell D-Tip S-Tip Cell Cell S-Tip You can develop specific markers for Diving tip calls and superficial tip cells. You can see what is or isnt expresssed in the different populations of cells. IsoB4 Laminin IsoB4 IsoB4 IsoB4 CLD5 IsoB4Laminin CLD5 IsoB4Laminin CLD5 IsoB4 Coll-IV Coll-IV IsoB4 IsoB4 Coll-IV IsoB4MFSD2A CLD5 IsoB4 Laminin IsoB4 S-Tip cells (P6) IsoB4MFSD2A CLD5 IsoB4 Laminin IsoB4 IsoB4 CLD5 IB4 CLD5 Del Toro R. et al, Blood, 2010 Zhao Q. et al, Cancer Res., 2018 Benz P.M. et al, Acta Physiol (Oxf)., 2020 Deckelbaum R.A. et al, Angiogenesis, 2020 IsoB4 CLD5 IsoB4 IsoB4MFSD2A ESM1 IsoB4 CLD5 IsoB4 IsoB4MFSD2A ESM1 IB4 CLD5 IB4ESM1 M2A IB4 D-Tip Cell Cell D-Tip IsoB4 CLD5 IsoB4 IsoB4MFSD2A ESM1 IB4 IB4CLD5 Lam IB4 M2A 10μm 25μm 25μm 25μm 100μm S-Tip Cell Cell S-Tip UMAP 2 S-Tip Cell Cell S-Tip UMAP 1 IB4 CLD5 Tip cell markers D-Tip Cell Cell D-Tip IsoB4MFSD2A CLD5 IsoB4 Laminin IsoB4 (P10) IsoB4 CLD5 10μm 10μm 10μm 25μm 25μm 25μm D-Tip cells IB4Coll-IV Lam IB4 CLD5 IB4 25μm 100μm 25μm IsoB4MFSD2A ESM1 IsoB4 10μm 25μm 10μm IsoB4MFSD2A ESM1 IsoB4 IsoB4MFSD2A ESM1 IsoB4 IB4 IB4ESM1 M2A IsoB4 CLD5 IB4 CLD5 Characterization of the specialized neuroretina D-tip cell WNT pathway BRB genes WNT/β-Catenin target genes these genes are all important Tip cell D Tip cell S Arterial ECs Capillary ECs Proliferative ECs Venous ECs -0.1 0.0 Decreased 0.1 Increased -0.10 -0.05 0.00 0.05 -0.2 -0.1 0.0 0.1 GSVA score TGFβ target genes Tip cell D 1 Muller Glia P6 Tip cell S Identity 0 Arterial ECs Capillary ECs Astrocytes P10 Venous ECs Immune Cells P6 0.1 GSVAscore 0.2 0 25 50 75 100 Ve gf a Tg fb Tg 1 fb Tg 2 fb 3 Proliferative ECs 0.0 Percent Expressed Astrocytes P6 Immune Cells P10 -0.1 −1 ll D ce Tip ll S ce s Tip EC ial ter s Ar EC ary s pill C Ca ve E i rat ECs life s Pro enou V avg.exp.scaled Muller Glia P10 Features (Alk5)Tgfbr1 Tgfbr2 Retina vascularization relies on tip-cell heterogeneity Zarkada G. et al, Dev Cell 2021 Ocular neovascular diseases Premature retinop athy Diabetic Retinopathy There is excess oxygen to baby’s brain, and angiogenegis will STOP because of this. THere will be vasoregression. at later stages, this inflammation will increase agniogenesis, and induce leaky blood vessels in to the retina Ideally, proliferative retinopathy’s treatment would selectively inhibit pathological angiogenesis while promoting retinal revascularization Ocular neovascular diseases Proliferative DR you should not have this on the fovea which can cause blind spots and extra bleeding Laser surgery for the eyes = Laser photocoagulation The procedure has risks, including: Accidental treatment of the central macula, which causes a worse blind spot Bleeding into the eye Damage to the retina from the laser scar, immediately or years later Abnormal blood vessels might grow back Ocular neovascular diseases – new anti-VEGF therapeutics Anti VEGF therapeutic (Aflibercept, Ranibizumab, Bevacizumab…) Proliferative DR Developped small molecules that bind receptos or trap VGEF ligands to block its signalling. Intravitreal injection: three injections at regular intervals of 4 weeks. Then, some patients will require further injections depending on the leakiness of the blood vessels (usually six weeks). VEGF inhibition in AMD: a success story It was a success!! The patients that were treament, dicovered their sight again Limitations: expensive, not all patients respond, concern regarding photoreceptor survival —> some patients develop resistance Can we target VEGFR2 downstream signaling pathways selectively? Objectif: Identifying new therapeutic targets for ocular neovascular diseases Challenges for the field: Ø Identifying new drugs to inhibit pathological angiogenesis with limited adverse effects on physiological angiogenesis. Ø Developing new drugs/strategies to facilitate the formation of functional blood vessels for tissue regeneration following ischemic injury. Approach: Ø Investigating and characterizing novel regulators of sprouting angiogenesis. Experimental models: Ø Utilizing genetic mouse models with temporally inducible, cell type-specific gene deletions using the Cre-lox system. Ø Developing novel therapeutic drugs tailored to specific targets. Ø Assessing retinal vascular phenotypes in developmental and pathological models. Amgioneurines: Endothelial cells express guidance receptors ,2 Rama, Dubrac et al., Nat Med 2015 Dubrac et al., Circulation 16 Zhang et al, Nat Comm 16 DCC Lu et al., Nature 2004 Koch et al., Dev Cell 2011 Brunet et al., JCI 2014 DCC Jones et al., Development 2008 Xu et al., J Cell Biol 2010 Bouvrée et al., Circ Res 2012 Aspalter, Gordon et al., Nat Comm 2015 Mouse model for ocular neovascular disease: Oxygen-induced retinopathy Room#air# making hypoxia enivronment realeasing a fuck to of VEGF, whcih causes many retinopathyies (high bleeding, leaking) P0# IB4 P12 no OIR 75%#oxygen# P7# Room#air# P12# IB4 P17# IB4 P12 OIR P17 OIR Mouse model for ocular neovascular disease: Oxygen-induced retinopathy Quantification IB4 IB4 P17 OIR Avascular area IB4 Neovascular tuft (NVT) Ideally, proliferative retinopathy’s treatment would selectively inhibit pathological angiogenesis (decrease NVTs) while promoting retinal revascularization (decrease avascular area) Inhibition of Slit2/Robo1&2 signaling prevents pathological ocular neovascularization If you block sliy1/2 exprtession, pathogenesis, you block Robo, whihc blocked the Slit signalling Gene deletion at P12 and analysis at P17 No compensation by VEGF signaling Can both pathways cross-talk and reinforce each other? Rama, Dubrac et al., Nat Med 2015