Exam 3: Dr. Olah Info

Cell injury

Cell injury

Cell is unable (or no longer able) to adapt to a stress, leads to detrimental physical/functional changes

Targets of cell injury

ATP production, cell membrane integrity, cytoskeleton, protein synthesis, DNA replication

cell injury outcomes

may be reversible if stressor is removed, irreversible can result in apoptosis or necrosis

Oxygen paradox

Oxygen is important for live but can be toxic in reduced forms

What is a radical vs free radical and what are characteristics of radicals?

molecule with a single unpaired electron, capable of independent existence; typically generated when a molecule gains an electron, usually oxygen or oxygen containing species (ROS) or nitrogen containing species (RNS), highly reactive

How are free radicals formed?

Natural process, normal reactions in healthy cells (enzymatic or non-enzymatic reactions), can form due to toxin exposure

Cell injury

Cell is unable (or no longer able) to adapt to a stress, leads to detrimental physical/functional changes

Targets of cell injury

ATP production, cell membrane integrity, cytoskeleton, protein synthesis, DNA replication

Superoxide radical

forms when molecular oxygen picks up another electron (3 total), some oxidant activity with limited membrane permeability

Source of intracellular ROS

ETC transports electrons, sometimes leak from a protein and are picked up by oxygen to form a superoxide radical

Generation of hydrogen peroxide and hydroxyl radical

a superoxide radical is converted to hydrogen peroxide via superoxide dismutase, easily generates the hydroxyl radical (extremely reactive and has limited ability to cross membranes)

Steps of hydroxyl radical generation from hydrogen peroxide

1. H2O2 may be converted to OH via the Fenton reaction, non enzymatic but requires iron or copper as electron donor

2. H202 may also react with a superoxide radical in the Haber-Weiss reaction (non-enzymatic)

Cytochrome P450 enzymes

heme-containing enzymes, metabolism of endogenous compounds, use electrons to activate oxygen for reaction, has “leaky” points (ROS)

NADPH Oxidase

important in cells of immune system, activated as response to invading pathogens, uses oxygen to generate free radicals to kill organism in conjunction with phagocytosis (respiratory/oxidative burst)

Xanthine oxidase

necessary for final steps of purine catabolism to uric acid, molecular oxygen is reduced to a superoxide radical

Monoamine oxidase (MAO)

metabolism or catecholamines in nerve terminals and other cells, generates H202

What environmental factors can increase ROS?

ionizing radiation (ultraviolet, x-rays), pollutants, cig. smoke, chemicals, drugs

Nitric oxide

free radial, important in widespread signaling molecule, vasodilator, neurotransmitter, usually at low concentrations that don’t cause damage

Protection from ROS

antioxidant enzymes, non-enzymatic antioxidants, repair processes, sequestration, drugs/supplements

Superoxide dismutase (SOD)

primary defense against oxidative stress, three isoforms that differ in location and regulation of expression but all use a metal ion in catalysis

Mn SOD

expressed in mitochondria, critical enzyme, levels may be increase by stress, expression is altered in various cancers

CuZn SOD

expressed in cytosol, Cu used in catalysis, Zn used in enzyme stability, not highly susceptible to regulation

Glutathione peroxidase

contains selenium, located in mitochondria and cytosol, handles H202 outside of peroxisome

Glutathione reductase

necessary to cycle glutathione disulfide (GSSG) back to reduced form (GSH)

Catalase

located in peroxisomes, widely distributed (liver/kidney), involved in beta-oxidation of long fatty acids, H2O2 production, and substrate metabolism

Nrf2

normally bound in cytoplasm by Keap1 (promotes Nrf2 destruction), released by Keap1 in response to oxidative stress

Curcumin

polyphenolic compound in turmeric, activates Nrf2

Vitamin e (alpha-tocopherol)

fat soluble vitamin, widely distributed throughout body (obtained via diet), mixture of tocopherols

lipid per-oxidation chain breaker

allows Vitamin E to donate an electron and stabilize its free radical intermediate

Vitamin C (Ascorbic acid)

regeneration of active form of vitamin E, may interact with free radicals directly (donates electrons)

Flavonoids

multiple compounds with a flavone backbone, may have multiple mechanisms (produce free radicals, free radical scavengers, inhibit Fenton reaction)

Lycopenes

linear unsaturated hydrocarbon, red pigment of plants

Antioxidant effects

quench free radicals, increase expression of antioxidant enzymes

Free radical induced damage

chain reactions are set off and extract electrons from other molecules (DNA, lipids, proteins)

Examples of DNA damage

Mispairing, strand breaks, excision of bases, crosslinking

Guanine and OH mediated modification

frequent site of OH attack where the 8-position of ring is hydroxylated, 8-OH-G is mutagenic and carcinogenic (can be measured in urine and used as a biomarker for oxidative stress)

Results of ROS-induced DNA damage

DNA damage detected, signaling proteins relay message, effector proteins/processes respond, DNA is repaired

Steps of base excision repair

Glycosylase excises the damaged base

Nuclease cuts the phospholipid backbone just before the missing base

Polymerase and ligase adds in a new base

What happens if DNA repair mechanisms fail?

cell replication may stop, cell may undergo apoptosis

Lipid Damage

site of attack is the lipids of membrane; mainly polyunsaturated fatty acids (allows for the free radical to extract H+), occurs in a chain reaction process (lipid peroxidation)

Steps of Lipid Peroxidation

1. Initiation- lipid becomes a lipid radical

2. Propagation- lipid radical reacts with oxygen to form lipid peroxyl radical

3. Degradation- Occurs when cells are damaged, produces malondialdehyde (carcinogenic, found in blood/urine)

4. Termination- Antioxidant may donate electron or radicals may recombine

End results of lipid peroxidation

cell injury due to destruction of membranes, other cell constituents affected (cross-links proteins)

Free radical-induced protein damage

free radical extracts H atom from amino acid residue, may target protein backbone or amino acid chain, usually cysteine and methionine

effects of protein oxidation

structure/location dictate whether or not the protein is targeted, cleavage of protein/chemical modification affects function

Types of defense for protein oxidation

1. Prevention- antioxidant enzymes

2. Specific defenses- enzymatic system may reverse oxidation of methionine

   1. Degradation of modified proteins by the proteasome- hydrophobic patches of the protein may be exposed and targeted for destruction

Ubiquitin-Proteasome System (UPS) function

Many proteins must be degraded by the cell, either normal protein turnover or a defense mechanism, often very protective, dysfunction can result in cell death or cancer

components of UPS

target protein (ubiquitin), E1/E2/E3 proteins(places ubiquitin proteins on substrate protein), proteasome(complex with regulatory regions can contains proteases in central region)

Cardinal signs of parkinsons (TRAP)

tremor-often observed at rest, “pill-rolling”

rigidity-increased muscle tone

akinesia-slowness, fatigue, interruption in movement

posture- stooped, loss of balance

pathology of parkinsons

loss of dopaminergic neurons in substantia nigra

lewy bodies- intraneuronal structures that lack membranes

sources of oxidative stress

aging, decreased MTC activity, dopamine metabolism

alpha-synuclein

major component of lewy bodies in insoluble fibrillar form, may have role in dopamine synthesis and/or vesicular transport

Benefits of ROS

immune defense-kills microorganisms

inflammation-signaling molecules to increase permeability and leukocyte migration

MAPKs activation-may promote proliferation and/or survival

Supply vs demand in adequate oxygenation

supply- tissue perfusion with oxygenated blood

demand- 90% of O2 used for oxidative phosphorylation in MTC

hypoixa

inadequate oxygenation to meet demands of tissue, usually results from decreases supply

ischemia

overall decreased blood flow to tissue, includes glucose and other nutrients but tissues are likely hypoxic

acute decreased O2 supply

high altitudes, CO poisoning, surgery, pulmonary (asthma attack), pathophysiologic thrombus (MI)

chronic decreased O2 supply

vascular disorders, pulmonary (COPD), heart failure, anemia

compensatory responses to hypoxia

increased respiratory rate (short term), increased synthesis of erythropoietin (long term)

Heterodimeric transcription factors

HIF-1 beta- stable in all O2 conditions

HIF-1 alpha- regulated by O2 levels

vasculogenesis

development of new vessels from precursor cells

angiogenesis

sprouting of new vessels from pre-existing vasculature, typically occurs at the capillary level, involves endothelial cells and sometimes pericytes if forming more fully developed vessels

schematic of angiogenesis

pericytes and extracellular matrix (ECM) are loosened

endothelial cell migration

endothelial cell proliferation

Role of matrix metalloproteinases

degrades the basement membrane of endothelial cells via proteolytic activity (enzymes that break down cells)

Process of endothelial cell migration

cells respond to attractive/repulsive signaling cues (involves receptor activation of small G proteins)

Process of endothelial cell proliferation

EC number must increase to make more vessels, enhanced by angiogenesis, slow turnover rate

Process of endothelial cell tube formation

represent proliferation and migration but also morphological changes that allow for branching and lumen formation

process of endothelial cell survival

proliferating/migrating ECs need protection during these processes, growth factors and their receptors are often anti-apoptotic

Angiogenic stimuli

hypoxia-most potent

receptor tyrosine kinases- growth factors receptors, VEGF

GPCRs-several involved

Vascular endothelial growth factor (VEGF)

secreted protein produced by many cell types, expressions/secretion stimulated by many factors, potent stimulus involved in angiogenesis (stimulates EC reponses)

VEGF receptors

all are RTKs, mostly associated with angiogenic responses on endothelial cells

Additional stages of blood vessel development

blood vessels need to be stabilized by surrounding pericytes

Diseases characterized by impaired blood flow (ischemia)

coronary/peripheral artery disease, diabetes

Limitations of angiogenesis therapies

incomplete understanding, multiple factors needed, difficult to deliver factors

Angiogenesis-dependent disease (progression of the disease)

ocular diseases, cancer, chronic inflammation

angiogenesis and cancer

tumors require vasculature b/c environment is often hypoxic so VEGF is released to promote vascular development (angiogenesis)

Targeting VEGF as a cancer treatment

inhibition of angiogenesis, combination therapy with anti-cancer drugs

VEGF-targeted anticancer drugs

Bevacizumab (avastin) and Sorafenib (Nexavar)

Inflammation

body’s response to injury, involves vascular/cellular responses to defend or repair

Causes of inflammation

infectious pathogen, foreign bodies, trauma, physical/chemical agents, tissue necrosis, hypersensitivity reactions

Influence of initiating stimulus on inflammatory response

intensity, duration, outcomes of response

examples of bad inflammation

‘itis (asthma, colitis, rheumatoid arthritis)

cardinal signs of inflammation

rubor (redness), swelling, calor (heat), dolor (pain), functio lasea (loss of function)

cytokines

proteins or peptides released by immune cells, activate cell surface receptors, involved in inflammatory process and cell signaling

vasodilation

increases blood flow to an area, result in calor and rubor, primarily observed in arterioles ex: histamine

paracellular leakage

junctions between ECs become loosened due to contraction of individual ECs

transcellular leakage

limited transport of proteins through ECs

causes of increased vascular permeability

EC injury (burn/frostbite), leukocyte mediated cell injury (infection), angiogenesis (chronic inflammation)

Extravasation of leukocytes

margination

tethering & rolling

integrin activation

firm adhesion

transmigration (diapedesis)

migration in interstitial tissue (chemotaxis)

margination

leukocytes accumulate on surface of blood vessel, occurs due to low blood flow

tethering & rolling

marginated leukocytes interact with endothelial cells and slow down, mediated by selectins (adhesion molecules)

types/structure of selectins

E-selectin: endothelial cells

P-selectin: platelets & endothelial cells

only expressed at surface of ECs that have been activated by cytokines or other inflammatory mediators

selectin-ligand interaction

ligands provide a region for proteins to bind, serves as the “tether”, fast on & fast of kinetics (rolling response)

integrins

located on leukocytes, involved in cell-cell or cell-matrix interactions, heterodimeric (alpha and beta domain)

Integrin activation

necessary for firm adhesion of leukocytes, smooth/round cell becomes flattened with extensions, use inside-out signaling and works with chemokines

Inside-out signaling

signaling comes from inside the cell and initiates a response outside the cell

Types of integrin receptor ligands on endothelial cells

Intercellular adhesion molecule (ICAM) and vascular cell adhesion molecule (VCAM)

How is surface expression of ICAM and VCAM upregulated?

by inflammatory cytokines and other signals, high affinity results in firm adhesion of leukocytes to endothelium

Outside-in signaling

used by ICAM and VCAM when integrin binds

Leukocyte transmigration (diapedesis)

leukocytes must leave the blood vessel(venule) to get to the damaged tissue without damaging the venule, usually paracellular(between adjacent ECs)

Paracellular transmigration

initiated as EC extends around adherent leukocyte, leukocytes may form pseudopods necessary for migration (same components as firm adhesion)