PI session 1

Question 1

What is cell death?

Answer 1

Irreversible severe cell injury that exceeds attempts at repair or adaptation induces cell death

Question 2

What are the two distictive forms of cell death?

Answer 2

• Apoptosis
• Necrosis

Question 3

Why does apoptosis occur?

Answer 3

• cell no longer needed by the body
• OR it is damaged beyond repair

Question 4

Why is there no inflammatory response associated with apoptosis?

Answer 4

• Dissolution of nucleus without perforation of cell membrane
• Prevents cell contents from leaking into extracellular matrix

Question 4

Why is there no inflammatory response associated with apoptosis?

Answer 4

• Dissolution of nucleus without perforation of cell membrane
• Prevents cell contents from leaking into extracellular matrix

Question 5

Cells destined to die activate intrinsic enzymes that degrade genomic DNA and nuclear and cytoplasmic proteins. These enzymes are called what?

Answer 5

Caspases

Question 6

What 2 pathways converge on caspase activation?

Answer 6

• Mitochondrial pathway (intrinsic) - mitochondrial membrane becomes more permeable and releases pro-apoptotic molecules
• Death receptor pathway (extrinsic) - activates inflammatory cascade

Question 7

What are the anti-apoptotic proteins in the intrinsic pathway?

Answer 7

BCL2, BCL-xL, and MCL1

Question 8

What are the pro-apoptotic proteins in the intrinsic pathway?

Answer 8

BAX and BAK

Question 9

What are the regulated apoptosis initiator proteins?

Answer 9

BAD, BIM, BID, Puma, and Noxa

sensors of cellular stress/damage & initiate apoptosis when activated

Question 10

What occurs in the intrinsic (mitochondrial) pathway of apoptosis?

Answer 10

• Growth factors and other survival signals stimulate BCL2, protecting cells from apoptosis
• When no signals are sent, or in the case of DNA damage or ER stress, BH3-only proteins (apoptosis initiators) are upregulated
• BH3-only proteins activate BAX and BAK which insert into mitochondrial membrane forming the permeability transition pore
• Results in cytochrome C leaking into the cytoplasm and binding to APAF-1 (apoptosis activating factor) and forms an apoptosome which binds CASPASE-9

Question 11

Explain the extrinsic (death receptor-activated) pathway with regards to Fas.

Answer 11

• When FasL binds to Fas, 3 or more molecules of Fas are brought together along the inner cell membrane, and their collective cytoplasmic death domains form a binding site for an adapter protein called FADD (Fas-associated death domain)
• FADD then binds to Caspase-8 (or caspase-10), activating the EXECUTIONER caspase sequence of the extrinsic path

Question 12

What is karyolysis?

Answer 12

• Basophilia of the nucleus fades
• Loss of DNA due to enzymatic destruction

Question 13

What is pyknosis?

Answer 13

• Nuclear shrinkage and increased basophilia
• Chromatin condenses into a ‘dense, shrunken basophilic mass

Question 14

What is karyorrhexis?

Answer 14

• Pyknotic nucleus undergoes fragmentation
• Within 1-2 days, the nucleus in the necrotic cell totally disappears

Question 15

What are the 6 patterns of tissue necrosis?

Answer 15

• Coagulative
• Liquefactive
• Gangrenous
• Caseous
• Fat
• Fibrinoid

Question 16

What occurs in coagulative necrosis?

Answer 16

• Denaturation of structural proteins and enzymes
• Shadow of dead cells/tissue persists for days
• Leukocytes eventually remove dead cells
• Classic example: infarcts (ischemic necrosis) in solid organs

Brain infarcts are an exception–> Liquefactive instead

Question 17

On histology slides, necrotic cells will lack what organelle?

Answer 17

Nucleus

Question 18

What are the gross appearance characteristics of infarcts?

Answer 18

• Firm
• Located near periphery of organ
• “Wedge shaped” (like triangle pointing toward center of organ)
• Pale/white, except for in lungs where it is red

Question 19

What is liquefactive necrosis?

Answer 19

• Dead cells completely digested into viscous liquid
• Examples: Abscess d/t bacterial infection; Infarct in brain/CNS
• If abundant inflammation (neutrophils): pus

Question 20

What is gangrenous necrosis?

Answer 20

• Not a specific pattern of cell death, but commonly used in clinical practice
• Coagulative necrosis involving a limb (lower leg)
• Superimposed bacterial infection attracting leukocytes and degradative enzymes causing liquefactive necrosis
• Combination causes so-called wet gangrene

Question 21

What is caseous necrosis?

Answer 21

• Grossly has a soft, pale, crumbly/friable “cheesy” look
• Buzzword for Tuberculosis
• Characteristic of a focus of inflammation called a granuloma

Question 22

What is fat necrosis?

Answer 22

• Focal areas of fat destruction
• Enzymes leak out of damage cells and liquefy membranes of fat cells in the peritoneum
• Typical of NECROTIZING PANCREATITIS - (abdominal emergency caused by leaking pancreatic lipases)
• Fat saponification: chalky white material - broken down lipid combines with calcium to make soap-like substance

Question 23

What is fibrinoid necrosis?

Answer 23

• Special form of vascular damage - Amorphous pink material (fibrin) associated with blood vessel walls
• Generally immune-mediated - Immune vasculitis (leukocytoclastic vasculitis, polyarteritis nodosa, etc)
• Only detected microscopically

Question 24

What is the ECM?

Answer 24

* A secreted network of proteins that surrounds cells and constitutes a significant proportion of any tissue in the body * Cell interactions within the ECM are critical for tissue development, healing, and maintenance of normal tissue architecture

Question 25

What are the two basic forms of ECM?

Answer 25

* Interstitial Matrix * Basement Membrane

Question 26

What is the interstitial matrix?

Answer 26

* 3-dimensional, amorphous, semi-fluid gel * Synthesized by mesenchymal cells (e.g., fibroblasts) * Occupies the spaces between stromal cells (in connective tissue) * Assists with support/scaffolding and in some tissues acts as a fluid that cushions tissue compression

Question 27

What is the basement membrane?

Answer 27

* Highly organized meshwork * Lies between epithelial cells and underlying connective tissue * Synthesized by overlying epithelial cells and underlying mesenchymal cells * Forms a flat, porous mesh that represents a specialized surface for cell growth

Question 28

What are the functions of the ECM?

Answer 28

* Mechanical support * Regulation of cell proliferation * Scaffolding for tissue renewal - **Disruption of the ECM prevents effective tissue regeneration and repair!** * Foundation for establishment of tissue microenvironments

Question 29

What are the components of the basement membrane?

Answer 29

* Type IV collagen * Laminin * Proteoglycan

Question 30

What are the components of the interstitial matrix?

Answer 30

* Fibrillar collagens * Elastin * Proteoglycan & Hyaluronan

Question 31

Fibrillar collagen does what? | types I, II, III, & V

Answer 31

Form linear fibrils that comprise a major proportion of connective tissue as well as in healing wounds and scars

Question 32

Nonfibrillar collagen does what? | type IV

Answer 32

Contributes to basement membrane structure, provides an anchor that maintains structure of some tissues, such as stratified squamous epithelium (e.g., skin)

Question 33

What is the function of elastic fibers?

Answer 33

Provide tissue recoil and return to baseline structure after physical stress

Question 34

What are water-hydrated gels? | comprised of?

Answer 34

* Provide compressibility to tissues, resilience, and lubrication * Reservoir for growth factors secreted into the ECM * Proteoglycans (glycosaminoglycans: ie- Keratan sulfate and chondroitin sulfate) are linked to hyaluronan. some are integral cell membrane proteins w/ roles in cell proliferation, migration, and adhesion * These proteoglycan – hyaluronan molecules attract sodium and water, producing a viscous, gel-like matrix * Present in skin, cartilage, vitreous humor, synovial fluid

Question 35

What is fibronectin?

Answer 35

* component of interstitial matrix * Provides the scaffolding for ECM deposition, angiogenesis, and re-epithelialization in healing wounds

Question 36

What is laminin?

Answer 36

* most abundant glycoprotein in the basement membrane * Connects cells to underlying basement membrane; modulates cell proliferation and differentiation

Question 37

What are integrins?

Answer 37

* a type of adhesion receptor, also known as cell adhesion molecules [CAMs] * Receptors that allow cells to attach to the adhesive glycoproteins (fibronectin, laminin) * Facilitate cell-cell adhesive interactions * Involved in signaling cascades that regulate cell locomotion, proliferation, shape, and differentiation

Question 38

What is tissue regeneration and when does it occur?

Answer 38

* Restoration of normal cells * Ability to regenerate is determined by (1) the ability of the cells to proliferate, and (2) the presence of tissue [adult] stem cells

Question 39

What is tissue scarring and when does it occur?

Answer 39

* Deposition of connective tissue * Occurs if (1) the injured tissues cannot regenerate, or (2) if the supporting structures (ECM) are too severely damaged to support regeneration

Question 40

With mild injury (damage to epithelium but not the underlying tissue), what type of tissue repair occurs?

Answer 40

**Regeneration**

Question 41

With severe injury (damage to connective tissue), what type of tissue repair occurs?

Answer 41

**Scarring**

Question 42

What is Labile tissue?

Answer 42

* continuously dividing; never enter G0 of cell cycle and divide rapidly with short G1) * *Examples: Hematopoietic cells, skin, and surface epithelia of the GI tract and genitourinary tissues* * Cells of these tissues are constantly being lost and replaced by (1) maturation from tissue stem cells and (2) proliferation of mature cells * Must preserve pool of stem cells for regeneration! * Most affected by chemotherapy!

Question 43

What is stable tissue?

Answer 43

* quiescent – G0 of cell cycle – but capable of dividing – enter G1 when stimulated * *Examples: Endothelial cells (blood vessels), smooth muscle cells, parenchymal cells of most solid tissues (liver, kidney, pancreas), lymphocytes* * **With the exception of the liver,** these cells have limited capacity to regenerate after injury * Liver has remarkable ability to regenerate after partial hepatectomy – (1) hepatocyte proliferation and (2) regeneration from “progenitor” cells in “canals of Hering” (adult stem cell niche!) * Healing ability is dependent on ability of these cells to proliferate

Question 44

What is permenant tissue?

Answer 44

* terminally differentiated; non-proliferative – G0 only) * *Examples: Neurons, cardiac muscle, skeletal muscle, red blood cells* * Injury to the brain, spinal cord, or heart is generally irreversible and results in a scar because this tissue cannot regenerate (very limited exceptions)! * Repair of these tissues is dominated by scar formation

Question 45

What are the steps in scar formation?

Answer 45

Within minutes, hemostasis occurs, followed by: 1. Inflammatory response (aka inflammatory phase of wound healing) 2. Cell proliferation and angiogenesis (aka proliferative phase) 3. Formation of granulation tissue (proliferative phase continued) - Collagen deposition and ECM protein synthesis 4. Remodeling of connective tissue and wound contraction (aka remodeling phase) 5. Acquisition of wound strength (remodeling phase continued)

Question 46

What are the mediators in step 1 (inflammation) of scar formation?

Answer 46

* Break-down products of complement activation; cytokines (from activated platelets) – function as chemotactic agents and recruit **(1) neutrophils** followed by **(2) macrophages** * **Growth factors** [GF] (from platelet plug, endothelial cells, other inflammatory cells) * **PDGF (platelets, macrophages)** - induces vascular remodeling, smooth muscle cell migration; stimulates fibroblast growth for collagen synthesis

Question 47

After clot formation (hemostasis), step 1 of scar formation (inflammation) is characterized by what?

Answer 47

* Increased vessel permeability and neutrophil migration into tissue * Inflammatory cells eliminate the injurious agents (such as microbes) * Macrophages clear debris, necrotic cells

Question 48

What occurs in step 2 of scar formation (proliferative phase)?

Answer 48

Several cell types proliferate and migrate to close the now clean wound * **Epithelial cells** – respond to locally released GF and migrate to the wound to cover it (skin: keratinocytes) * **Endothelial cells and pericytes** – undergo **angiogenesis** (proliferation to form new blood vessels) * **Fibroblasts** – proliferate and migrate to the wound; lay down collagen fibers that form the scar; deposit ECM proteins

Question 49

What is angiogenesis?

Answer 49

the process of new blood vessel development from existing vessels

Question 50

What growth factors are involved in angiogenesis?

Answer 50

**VEGF (Vascular Endothelial GF)** * Stimulates migration and proliferation of endothelial cells * Promotes vasodilation (stimulates production of nitric oxide [NO]) **FGF-2 (Fibroblast GF-2)** * Stimulates endothelial cell proliferation * Promote migration of macrophages and fibroblasts to wound * Stimulate epithelial cell migration to cover wound Angiopoietins 1 and 1 (Ang 1 and Ang 2) * GFs that promote angiogenesis and structural maturation of new vessels

Question 51

What occurs in step 3 of scar formation (formation of granulation tissue)?

Answer 51

Fibroblasts continue to proliferate, deposit loose connective tissue (mediator: TGF-beta), and synthesize ECM proteins forming granulation tissue

Question 52

How is granulation tissue characterized?

Answer 52

* Proliferation of fibroblasts * New, thin-walled, delicate vessels (angiogenesis) * Loose ECM, often with admixed inflammatory cells (mostly macrophages) * Progressively fills the wound; the amount formed depends on size of tissue defect, and intensity of inflammation

Question 53

What occurs in step 3 of scar formation (deposition of connective tissue, ECM)?

Answer 53

* Granulation tissue is progressively replaced by the deposition of **collagen** * **Collagen is critical for the development of wound strength** * Mediated by locally produced cytokines and GF: **PDGF**, **FGF-2**, **TGF-beta**

Question 54

Explain the role of TGF-beta in step 3 of scar formation.

Answer 54

* Potent fibrinogenic agent: most important cytokine for the synthesis and deposition of connective tissue proteins * Produced by most of the cells in granulation tissue, including macrophages * Also acts as an anti-inflammatory cytokine by inhibiting lymphocytes and the activity of other leukocytes

Question 55

What is the end result of step 3 of scar formation?

Answer 55

Highly vascularized granulation tissue eventually transforms into a pale, largely avascular scar * Amount of connective tissue increases; deposition of ECM increases * In contrast, there is progressive vascular regression * Some fibroblasts acquire features of smooth muscle cells called **myofibroblasts** - cause scar contraction

Question 56

What occurs in step 4 (remodeling of connective tissue) of scar formation?

Answer 56

**Matrix metalloproteinases (MMPs)** * Dependent on metal ions (e.g., zinc) for their activity * Essential for remodeling * Produced by various cell types (fibroblasts, macrophages, neutrophils, some epithelial cells) * **Mediate degradation of collagens and other ECM components** * Activity is tightly regulated * Produced as inactive precursors that must be activated by proteases that are expected to be present only at site of injury * Can be rapidly inhibited by specific **tissue inhibitors of metalloproteinases (TIMPs)**, produced by most mesenchymal cells

Question 57

What occurs in step 5 of scar formation?

Answer 57

* Excess of collagen synthesis over collagen degradation (due to cross-linking of collagen fibers and increased fiber size) * **Type III collagen is replaced by type I collagen (mediated by fibroblasts)** * Results in increased tensile strength of tissue