L13 - Vascular Cell Biology 2024 - Mujica.pdf

Blood Vessels as Specialized Organs Patricio E. Mujica, Ph.D. [email protected] MSB H655 Objectives 1. Describe the structural organization of the blood vessel wall and correlate it with its functional properties across vascular beds. 2. Compare and contrast the functions of different type...

Blood Vessels as Specialized Organs Patricio E. Mujica, Ph.D. [email protected] MSB H655 Objectives 1. Describe the structural organization of the blood vessel wall and correlate it with its functional properties across vascular beds. 2. Compare and contrast the functions of different types of blood vessels. 3. Explain the role of vascular smooth muscle cells as contractile / proliferative cells, and the mechanisms governing their function. 4. Explain the role of vascular endothelial cells as regulators of vascular integrity, and the mechanisms involved in their function. 5. Explain the role of nitric oxide as a central regulator of endothelial function. 6. Compare and contrast the two main forms of neovessel formation and explain the relevance of angiogenic processes for homeostatic and pathologic conditions. 2 Why is vascular health important? People die of dysfunction of blood vessels. Cause of death in developed countries Understanding of molecular / cellular mechanisms of blood cell function is vital 3 for public health and to improve diagnosis and therapeutics development 1. Introduction: Structural and Functional Diversity of Blood Vessels 4 Heart and blood vessels –vital organs evolutionarily conserved ü Blood vessels are found throughout the body, and their main function is to establish a delivery and exchange system for oxygen and nutrients to tissues. ü Blood vessels also play a role in controlling blood pressure. ü Regulation of inflammation and immune responses ü Many other important organ-specific functions ü Metabolic regulation in the adipose and skeletal muscle. ü Specialized blood-tissue barriers: brain, testes, lymphoid organs, etc. ü Artery, vein, lymphatic vessels Five main types of blood vessels: arteries, arterioles, capillaries, venules and veins. Arteries exhibit vasoconstriction in response to a1 adrenergic stimulation or local vasoconstrictors such as Ang-II, endothelin-1. Vasodilation mediated by endothelium-derived nitric oxide (NO) in response to acetylcholine, bradykinin, prostaglandins (PGI2), and others. Deliver blood to capillaries; also capable of constricting or dilating à metabolic or myogenic regulation controls perfusion of downstream capillary beds Very thin walls allow exchange of nutrients, waste and gases between blood and body tissues. Collect blood from capillaries and drain into veins Carry blood back to the heart. They may contain valves 6 Five main types of blood vessels: arteries, arterioles, capillaries, venules and veins. 7 How the blood vessels perfuse the tissues, and supply oxygen and nutrients. 8 The vascular wall is made of three layers and several cell types: structure/function relationship 1. Tunica externa or adventitia — fibroblasts and ECM 2. Tunica media — vascular smooth muscle cells 3. Tunica intima — endothelial cells and pericytes 9 2. Vascular Smooth Muscle Cells: Mechanisms that Control Vascular Tone and and Proliferation 10 Vascular smooth muscle cells (VSMCs) 11 Vascular smooth muscle cells (VSMCs) Vascular smooth muscle cells (VSMCs) During contraction the filaments slide together making cell rounder Two Mechanisms for the Regulation of VSMC Contraction 1. Calcium-dependent pathway Calcium entry into VSMCs can be triggered by several stimuli: - Factors activating Gq-coupled signaling - Angiotensin-II - Alpha1 adrenoceptor agonists - Cell depolarization - Opening of L-type Ca2+ channels - Cell stretch - Piezo1/2, TRPV4 mechanosensitive channels - Pro-inflammatory agents - Platelet-activating factor 14 Two Mechanisms for the Regulation of VSMC Contraction 2. Rho-ROCK pathway Rho GTPases operate as molecular switches ü Bind to and hydrolyze GTP ü GTP-bound form is active ü GDP-bound form is inactive ü Switching between GDP-bound and GTP-bound forms is regulated by enzymes: ü GEFs (guanine exchange factors) trigger release of GDP and binding to GTP ü GAPs (GTPase-activating proteins) induce GTP hydrolysis into GDP In VSMCs: ü Activation of G12/13-coupled signaling activates p115-RhoA GEF ü RhoA-GTP binds to and activates Rhodependent kinase (ROCK) 15 Two Mechanisms for the Regulation of VSMC Contraction 2. Rho-ROCK pathway Nunes and Clinton Webb (2021) PMID: 32970516 Pharmacological Inhibition of ROCK by Y-27632 Leads to Vessel Relaxation Spontaneously hypertensive rats Structure of Y-27632 % Relaxation Rat aortic rings; contraction elicited by KCl or PE application for 1 hour. Y-27632 applied at increasing concentrations 50.9 mM KCl, 1h 1 uM phenylephrine, 1h Blood pressure change (mmHg) Renal hypertensive rats DOCA-salt rats Control Wistar rats Y-27632 concentration (-log M) Time (h) Uehata et al (1997), Nature, 389: 30 Dose-dependent effect of Y-27632 on 17 blood pressure of hypertensive rats. Smooth Muscle Relaxation Depends on Myosin Light Chain Dephosphorylation by MLCP VSMC Relaxation requires two events: 1. Decrease in intracellular calcium concentration 2. Myosin light chain dephosphorylation #1 is achieved through: - Calcium pumping back into SR by SERCA - Calcium extrusion out of the cell by PMCA and NCX #2 is achieved through: - Reducing MLCK activity - Increasing MLCP activity In resistance vessels (medium and small arteries, and most arterioles which control blood pressure and tissue perfusion), #2 is the direct result of endothelial NO production - we’ll talk about this mechanism later VSMCs phenotypic switching and atherosclerosis/arteriosclerosis atherosclerosis arteriosclerosis Atherosclerotic plaque Neointima (Proliferated VSMCs) 19 Rho/ROCK Signaling is Involved in the Pathogenesis of Arteriosclerosis Rat carotid artery model of neointimal formation: balloon injury Phosphorylation of MYPT1 and MLC20 are indicators of ROCK activity p27kip1 is an inhibitor of cyclin-dependent kinases, which regulate cell proliferation 20 3. Endothelial Cells Regulate Vascular Function and Integrity 21 Vascular Endothelial Cells (ECs) ü Form a mono-cellular layer (called the endothelium) lining the lumen of all blood vessels and the surfaces of the heart chambers (the endocardium) ü Constitute a large “organ”: Account for about 1 kg of an adult human body, which is on par with the liver. (If spread out, all the endothelium in an adult would take up the area of 8 tennis courts) ü In the larger vessels (veins and arteries), the endothelium forms the blood vessel wall along with VSMCs, elastic fibers, and adventitia. ü In the capillaries, however, the endothelium makes up the entirety of the blood vessel wall. ü Endothelial cells are organotypic: their structure and functional properties depend on the organ/tissue where they are found 22 Vascular Endothelial Cells (ECs) ü Quiescent EC monolayer provides a selectively permeable barrier between the blood and tissues. Substances and leukocytes (white blood cells) in circulation move in and out in a regulate manner across EC barrier to where they are needed. Achieved by transcellular (i.e., transporter) and paracellular (i.e., cell-cell junctions, EC contraction) mechanisms. ü Upon tissue injury or in response to needs for more oxygen and nutrients supply (exercise, hypoxia/ischemia in diseases), ECs become proliferative/migratory and forms new blood vessels (a function called sprouting angiogenesis). 23 Vascular Endothelial Cells (ECs) Canine coronary postcapillary venule 24 Nitric Oxide is the Central Regulator of Endothelial and Vascular Homeostasis ü Nitric Oxide (NO) is a gaseous free radical and the central player of EC function, that 1. Relaxes VSMCs and dilates blood vessels to control blood flow and blood pressure 2. Prevents platelets from sticking to vessel walls (anti-coagulation) 3. Regulates (suppresses) inflammation by controlling the expression of adhesion molecules (E-selectin, VCAM1, ICAM1). ü In ECs, NO is produced by the endothelial nitric oxide synthase (eNOS); a highly regulated enzyme with a complex dimeric structure. Förstermann and Sessa (2012), Eur Heart J 33(7):829 25 eNOS Function Depends on its Coupling ü Lack of eNOS cofactors leads to enzyme uncoupling and production of strong oxidizing free radicals that damage cellular components and leads to cell death ü eNOS knockout mice show increased blood pressure, propensity to manifest exaggerated atherogenesis, and accentuated vulnerability to cardiac and brain ischemia (aggravated infarction) due to reduced collateral blood flow. ü Humans with atherosclerosis, diabetes, or hypertension often show impaired NO pathways. Endothelial NO Elicits cGMP Production and Relaxation of VSMCs GPCR: G-protein coupled receptor eNOS: endothelial nitric oxide synthase sGC: soluble guanylyl cyclase cGMP: cyclic guanosine monophosphate PKG: Protein kinase G / cGMP-dependent kinase MLCP: myosin light chain phosphatase MLCK: myosin light chain kinase Da Silva et al (2021) https://doi.org/10.3390/biology10101041 ACh Bradykinin Histamine Endothelial Rho-ROCK Signaling Contributes to Hypertension Development by Inhibiting eNOS ü EC Rho/ROCK has been shown to inhibit eNOS. ü ROCK inhibitor Y-27632 restores eNOS expression and activity, downregulated in diseased conditions. Sawada and Liao, ANTIOXIDANTS & REDOX SIGNALING, 2013 28 Microvascular permeability is determined by the barrier properties of the endothelium Lowest permeability Brain (blood-brain barrier) Lung Skeletal Muscle Heart Large intestine Kidney (glomerulus) Spleen Highest Liver (sinusoids) permeability 29 Agents that increase microvascular permeability are pro-inflammatory mediators ü Bradykinin, histamine, platelet-activating factor, vascular endothelial growth factor ü Act on post-capillary venules Control 10–6 M Histamine, 3’ (topical) Hamster cheek pouch, intravital microscopy; FITC-Dextran 150 kDa (albumin mimic)30 eNOS Trafficking and Localized NO Signaling Regulates Vascular Barrier Function and Controls Inflammation WT eNOS–/– Basal 5min 10min 15min 30min 8cPT: Epac-selective cAMP agonist Control PAF PAF + 8cPT eNOS 31 eNOS Trafficking and Localized NO Signaling Regulates Vascular Barrier Function and Controls Inflammation 32 Nepali et al., 2023, Am J Physiol 4. Angiogenesis: A Complex Process 33 ECs Drive the Formation of New Blood Vessels From Existing Ones ü EC functions can be broadly classified into two types of activities: ü Quiescent ECs line the vascular wall and provide a barrier to solutes, regulate nutrient transport, and maintain bloodstream homeostasis. ü In response to stimuli such as a wound, ECs escape dormancy, rapidly proliferate, and invade hypoxic and ischemic tissues in order to reestablish vascular support, a process known as angiogenesis. 34 Two Modalities of Neovessel Formation Sprouting angiogenesis ~75% Driven by local ECs: dedifferentiation, migration, proliferation Vasculogenesis ~25% Driven by endothelial progenitor cells from the bone marrow (CD34+) 35 Diabetic foot suffers impaired collateralization Critical limb ischemia (CLI) occurs when blood flow to limbs is reduced or interrupted for a long period of time Diminished angiogenesis Diminished vasculogenesis Vascular Ischemia occlusion Foot ulcer Amputation Under normal conditions, ischemic tissues release pro-angiogenic signals that lead to vessel collateralization Diabetic pathophysiology impairs this process à tissues cannot regain oxygen supply Affects 10% of patients Leading cause of limb amputation (1 leg / 30 sec) Poor prognosis (5-yr survival 32%) 36 Vascular Endothelial Growth Factors (VEGFs) and Their Receptors Are Key Mediators of Angiogenesis ü Angiogenesis is orchestrated by the interplay of multiple signals and receptors: ü VEGF – VEGFRs ü Ephrin – Eph receptors ü Angiopoietin – Tie ü Delta – Notch system ü VEGFs and VEGFRs regulate both vasculogenesis and angiogenesis – + ü VEGF-A stimulates angiogenesis, strongly increases vascular permeability, and stimulates cell migration in endothelial and myeloid cells ü Lymphangiogenesis, the sprouting of new lymphatic blood vessels, is also regulated by the VEGF system. Shibuya M, Genes Cancer. 2011 37 Angiogenesis Is Critical For Tumor Growth Liu et al (2023) PMID: 37169756 38 VEGF: A Double Sword in Translational Research ü Anti-VEGF and anti-VEGFR drugs are widely used for the treatment of major solid tumors. Ø Anti-VEGF-A neutralizing antibody Ø Multi-kinase inhibitors ü The clinical efficacy of these medicines has been well evaluated; however, none of them provide a complete cure for cancer patients. ü There is a need to more completely characterize the molecular basis of these phenomena to develop better anti-angiogenic therapies. ü On the other hand, VEGFs have pro-angiogenic potential for the maintenance of various tissues at physiological levels Ø High potential for the treatment of ischemic diseases. Ø Downside: VEGF gene therapy failed for critical limb ischemia VEGF resistance? ü The utility of VEGF family members in pro-angiogenic medicine, together with the possible side effects, should be characterized in more detail for clinical applications. 39 VEGF Resistance May Be Responsible For Impaired Neovessel Formation in Ischemic Disease 40 Notch Signaling Regulates Angiogenesis and Is Increased in Pathological States 41 Notch Signaling Regulates Angiogenesis and Is Increased in Pathological States Confers VEGF resistance and angiostasis Roles of Notch in VEGF resistance of ischemiainduced angiogenesis in DM Blood 2006 Nat Med 2008 Roles of Notch in diabetic nephropathy Cao et al. Biomaterials 2010 42