PATH

Obj 1

Effects of Inflammation:

1. Rubor- redness
2. Tumor- swelling
3. Calor- heat
4. Dolor- pain
5. Function laesa- loss of function
Rubor, tumor and calor found in acute inflammation are caused by the increased blood flow (arteriole
dilation) and edema (invasion of tissue by neutrophils)

Dolor is a product of the toxic metabolites and proteases that the leukocytes release in response to the
microbe. Prostaglandins, neuropeptides, and cytokines all contribute to the local pain (dolor)

Obj 2

Stimuli that trigger an acute inflammatory response (acute- fast onset, short duration)

1. Infection (bacterial, viral, fungal, parasitic) and microbial toxin release

a. TLRs detect these microbes and trigger signal pathways to inflammation
2. Tissue necrosis (ischemia, trauma, physical/chemical injury)
a. Molecules released by necrotic cells that trigger inflammation:
i. Uric acid
ii. ATP
iii. HMGB-1 (a DNA-binding protein)
iv. DNA (when released in cytoplasm instead of kept in nucleus)
v. Hypoxia (by HIF-1α, hypoxia induced factor, which activates VEGF, which
increases vascular permeability)
3. Foreign bodies (splinter, dirt, sutures)
a. Cause traumatic tissue injury or carry microbes
4. Immune reactions (hypersensitivity reactions)
a. Directed against self antigens  autoimmune disease
b. Excessive reactions against environmental substances or microbes

Obj 3

Vascular changes in acute inflammation:

1. Vasodilation of arterioles, then opening of new capillary beds in the area  increased blood
flow (the cause of heat and redness)
a. Induced mainly by histamine and NO on vascular smooth muscle
 2. Microvasculature permeability increased, allowing flow of exudate into tissue; due to:
a. Contraction of endothelial cells (by histamine, bradykinin, leukotrienes)  increased
interepithelial spaces; usually rapid and called “immediate transient response,” but can
be delayed, “delayed prolonged leakage”
b. Endothelial injury  endothelial necrosis, detachment
c. Transcytosis (increased transport of fluids/proteins through endothelial cell)
3. Slower blood flow, concentration of red cells, increased viscosity of blood  dilation of small
vessels that are packed with slow-moving red cells = stasis/vascular congestion
a. Producing localized redness
4. Neutrophils accumulate along endothelium, migrate through into tissue…

Exudate- fluid with high protein content, cellular debris, which leaks into interstitial tissue. Implies an
increased permeability of capillaries and thus an inflammatory response

Transudate- fluid low in proteins, little/no cellular debris; results from osmotic or hydrostatic pressure,
not vascular permeability

Obj 4

Role of endothelial cells in inflammation:

- Endothelium is activated during inflammation and can bind leukocytes, assisting their exit from
the blood vessels
- Secrete cytokines, which act as chemoattractants for neutrophils
- Express E-selectin and ligands for L-selectin to enhance attachment of leukocytes while “rolling”
- Express integrins, promoting a stronger adhesion to leukocytes as they roll

Leukocyte transmigration: migration of the leukocyte through the endothelim (aka diapedesis)

- Occurs mainly in post-capillary venules

- Stimulated by chemokines
- PECAM (platelet endothelial cell adhesion molecule) in endothelial junction to promote
migration
- Leukocytes secrete collagenases to pierce the basement membrane and enter the extravascular
tissue, then continue migrating toward the chemokines at the site of injury

Obj 5

Leukocyte activation, phagocytosis, killing of microbes:

1. Signaling pathway triggered in leukocytes  increase in cytosolic ca2+  activation of PKC and
PLA2 (phospholipase A2) = active leukocyte
2. Recognition/ attachment of particle to be ingested by leukocyte
a. Enhanced by opsonization
 3. Engulfment of the particle, formation of phagocytic vacuole
a. Using pseudopods, pinching off plasma membrane, forming a vesicle (phagosome)
which fuses with a lysosome  phagolysosome
4. Killing/degradation of ingested material
a. Respiratory burst: NADPH oxidase  ROS  H202  myeloperoxidase  HOCl (toxic)
b. Also Reactive Nitrogen Species  NO  OONO radical (peroxynitrite)

Obj 6

Acute inflammatory mediators:

- Vasoactive amines
o Histamine-
 stored in mast cell granules, basophils, and platelets
 released by degranulation in response to physical injury, antibody binding,
complement…
 causes dilation of arterioles, increases permeability of venules, produces
interendoepithelial gaps
o Serotonin-
 Stored in platelets (and some neuroendocrine cells in the GI)
 Released when platelets aggregate after contacting collagen, thrombin, ADP,
antigen/antibody complex
 Causes coagulation
- Arachidonic acid metabolites (aka eicosanoids)
o Prostaglandins
 Produced by mast cells, macrophages, endothelial cells… by cyclooxygenases
 Cause vasodilation, pain, and fever
o Leukotrienes
 Produced by lipoxygenases and secreted by leukocytes
 Chemoattractants, leukocyte adhesion, increased vascular permeability and
vasoconstriction
o Lipoxins
 Produced by neutrophils using lipoygenase
 INHIBIT inflammation, by inhibiting recruitment of leukocyte, chemotaxis, and
adhesion to endothelium
- Reactive Oxygen Species
o Release extracellularly from leukocytes after exposure to microbes, chemokines, immune
compleses, phagocytosis, by the NADPH oxidase complex
o Increase expression of chemokines, cytokines, endothelial leukocyte adhesion molecules 
amplifies inflammation
- NO
o By endothelial cells, macrophages, some neurons in brain
 Induces cyclic GMP  relaxation of vascular smooth muscle ( vasodilation)
- Cytokine (TNF and IL-1)
o Produced by macrophages, stimulated by endotoxin, immune complexes, physical injury)
o In endothelium- induced activation (expression of adhesion molecules, chemical mediator
synthesis, matrix remodeling enzymes)
 o IL-1- controlled by inflammasome (multi-protein complex that responds to microbial stimuli
and dead cells  activates proteases  cleave active cytokines
- Chemokine
o Produced by macrophages, endothelial cells, T lymphocytes, mast cells
o Bind GPCRs and recruit (chemoattractant for) leukocytes to inflammation, migration of cells
to normal tissue
- Leukocyte granule components
o Acid proteases- degrade bacteria/debris within the phagolysosome
o Neutral proteases- degrade extracellular components (collagen, basement membrane,
fibrin, elastin, cartilage), cleave complement proteins (C3 and C5)
o Elastase- degrade virulence factors of bacteria
o Acid hydrolase, collagenase, phospholipase, plasminogen- for chronic inflammation…
- Complement
o Inactive form in plasma- activated by cleavage of C3 (classic, alternative, lectin pathway)
 C3, C5 (somewhat C4a) stimulate histamine release  increase vascular
permeability, vasodilation = anaphylatoxins
 C3b and iC3b act as opsonins  phagocytosis
 MAC (membrane Attack Complex) makes cells permeable to water  cell lysis
- Coagulation system
o Hageman factor (factor XII)- synthesized in liver, activated by negative charge (when
vascular permeability allows proteins into tissue…)
 Kinin system- produces vasoactive kinins
 Clotting system- forms thrombin  inflammation
 Fibrinolytic  plasmin, degrades fibrin  fibrinopeptides  inflammation
 Complement system  anaphylatoxins

Obj 7

- Leukocyte adhesion deficiency type 1- mutation in integrins

- Leukocyte adhesion deficiency type 2- mutation in ligand for selectin
- Chronic granulomatous disease- decreased oxidative burst, mutation in phagocyte oxidase
membrane component (x-linked) or cytoplasmic component (autosomal recessive)
- Chediak-Higashi syndrome- decreased leukocyte function because of mutation affecting protein
involved in lysosomal membrane traffic; defective fusion of phagosomes and lysosomes in
phagocytes

Obj 8

Termination of acute inflammatory response:

- Partially because mediators of inflammation are produced in rapid bursts, only as long as
stimulus lasts, have short half lives, and are degraded after their release
- Also due to stop signals ( switch in type of arachidonic acid produced, switch from leukotrienes
to lipoxins, liberation of TGFβ and IL-10, production of resolvins and protectins)

Outcomes of acute inflammation:

 - Complete resolution- injury is limited and short-lived
- Healing by connective tissue replacement (fibrosis)- tissues can’t regenerate or too much fibrin
exudation
o Connective tissue grows into damaged area, converts damage into mass of fibrous tissue
= organization
- Chronic inflammation- acute inflammatory response cannot be resolved
o Pneumonia, peptic ulcers

Gross/Microscopic appearance of acute inflammation patterns: (for pictures, see Robbins pgs. 67-69)

- Serous inflammation- effusion (outpouring and accumulation) of fluid in peritoneal, pleural, and
pericardial cavities
- Fibrinous inflammation- larger vascular permeability  fibrin deposited in EC space of body
cavities
o Eosinophilic meshwork of threads
- Suppurative/purulent inflammation; abscess- production of pus
o Acute appendicitis
o Abscess contains neutrophils, cellular debris, surrounded by congested blood vessels
- Ulcer- sloughing of inflamed necrotic tissue (mucosa of mouth, stomach, intestine,
genitourinary, and skin, subcutaneous tissue of lower extremities)
o Peptic ulcer- shows crater with acute inflammatory exudate in the base

Obj 16

Lymph nodes: (for pictures, see Robbins pg. 596)

 - At rest: discrete encapsulated structures containing well-organized B-cell and T-cell zones,
contain many phagocytes and APCs
- Upon activation of immune cells: primary follicles enlarge, pale-staining germinal centers,
hyperplasia of paracortical T-cell zone
-
Lymphadenitis- inflammation of a lymph node, caused by, most commonly, bacterial infections from
streptococcal and staphylococcal bacteria. also common in infectious mononucleosis, cytomegalovirus
infection, toxoplasmosis, brucellosis, secondary syphilis, and disseminated histoplasmosis.

Acute Nonspecific Lymphadenitis- in cervical, axillary, inguinal, mesenteric regional lymph nodes

- Nodes are swollen, gray-red, engorged

- Large reactive germinal centers containing mitotic figures
- Macrophages contain debris from necrotic cells
- Centers of follicles may be necrotic
- Entire node may be filled with pus
- Endothelial cells undergo hyperplasia

Chronic Nonspecific Lymphadenitis

- Follicular hyperplasia- stimuli that activate humoral immune response

o Large oblong germinal centers, surrounded by small resting naïve B cells
o Preservation of lymph node architecture
o Variation of shape/size of follicles
o Mitotic figures, phagocytic macrophages, recognizable light/dark zones
- Paracortical hyperplasia- stimuli that activate T-cell response
o Activated T-cells 3x normal size
o Open chromatin
o Several nucleoli
o Hypertrophy of sinusoidal/vascular endothelial cells
- Sinus histiocytosis (aka reticular hyperplasia)
Increase in number/size of cells that line lymphatic sinusoids

14.27

How control of the cell cycle, proliferative rate, differentiation, and apoptosis modify tissue repair
reactions

Size of a population is determined by rates of cell proliferation, differentiation, and apoptosis

Proliferative Rates

 Largely controlled by signals from env that stimulate or inhibit proliferation

 Tissues divided into 3 groups based on proliferative activity
o Continuously dividing (labile): proliferate throughout life (epithelia of skin and GI,
mucosa of excretory ducts of body glands, bone marrow and hematopoietic tissue cells)
o Quiescent (stable): low level of replication (parenchymal cells of liver kidneys, pancreas,
mesenchymal cells of fibroblasts and smooth muscle, vascular endothelial cells,
lymphocytes, other leukocytes)
o Nondividing (permanent): cannot divide postnatally, neurons and cardiac muscle cells

Cell Cycle Control

 Cell replication stimulated by GFs or signaling from ECM components through integrins
 Cell cycle has a central role in maintaining tissue homeostasis and regulating regeneration and repair
o So neds multiple controls and redundancies (especially at G1/S interface)
 After passing the restriction point, cells are irreversibly commiteed to DNA replication
 Progression through cell cycle is regulated by cyclins and CDKs
o These are inhibited by CDKIs
 There are checkpoints that survey fro DNA or chromosomal damage
14.28

Define and compare the types of adult and embryonic stem cells and summarize thyeir roles in
maintaining tissues

Stem Cells: self renewal, can generate differentiated cell lineages, must be maintained during an
organisms lifetime to give rise to different cell lineages (maintained by obligatory asymmetric
replication- 1 daughter retains self renewal ability while other differentiates- or stochastic
differentiation- balance between divisions that generate 2 stem cells and 2 differentiating cells)

A change in differentiation of a cell from 1 type to another = transdifferentiation

This capacity= developmental plasticity

Embryonic Stem cells

 Pluripotent- can generate all tissues of body

o Pluripotent gives rise to multipotent- more restricted developmental potential- gives rise to
differentiated cells
 From the inner mass of blastocyst in embryonic development
Adult Stem Cells/Somatic Stem Cells

 More restricted capacity

 In skin, lining of gut, cornea, hematopoietic tissue (maybe in liver, pancreas, adipose tissue)
 Reside in niches- niche cells generate/transmit stimuli that regulate stem cell self renewal and
generation of progeny cells
 Divide slowly in most tissues
 Generate rapidly dividing cells (transit amplifying cells) that lose capacity to self perpetuate and
become progenitor cells
 There is little evidence that transdifferentiation of HSCs (hematopoietic stem cells) contributes to
tissue renewal in normal homeostasis or tissue regeneration and repair after injury

Induced Pluripotent Stem Cells (iPS cells)

 Differentiated cells can now be reprogrammed into pluripotent cells (similar to ES cells) by
transduction of genes encoding ES cell TFs
o TFs: Oct3/4, Sox=2, c-Myc, Klf4)
 Transfer nucleus of adult differentiated cell into an enucleated oocyte
 Inefficient and inaccurate (due to histone methylation-> improper gene expression)
 Can generate cells from enodermal, mesodermal, and ectodermal origin

Stem Cells in Tissue Homeostasis

 Bone Marrow
o Hematopoietic Stem Cells: generate all of blood cell lineages, reconstitute bone marrow
after depletion
o Marrow Stromal Cells: multipotent, can generate chondrocytes, osteoblasts, adipocytes,
myoblasts, and endothelial cell precursors depending on which tissue they migrate to, they
migrate to injured tissues and generate stromal cells, DO NOT seem to participate in notmal
tissue homeostasis
 Liver
o has stem cells/progenitor cells in canals of Hering
o can give rise to ocal cells (bipotential-> hepatocytes and biliary cells)
o funx as secondary/reserve and activated only when proliferation is blocked
 Brain
o neurogenesis from neural stem cells (NSCs) occurs in brain (so new neurons can be
generated)
o NCSs can generate neurons, astrocytes, and oligodendrocytes in subventricular zone and
dentate gyrus
o We do not know if new neurons are integrated into neural circuits under pathological
conditions
 Skin
o Located in
 hair follicle bulge: contribute to replenishment of surface epidermal cells ater skin
wounding but not in normal homeostasis (activation regulated by Wnt pathway and
inhibition of signals from BMP (bone morphogenic protein) system)
 interfollicular areas of surface epidermis: generate differentiated epidermis
 sebaceous glands
 Intestinal epithelium
o Stem cells in crypts (monoclonal) regenerate the crypts in 3-5 days
o Regulated by Wnt and BPM pathways
 Skeletal and cardiac muscle
o Skeletal muscle myocytes do not divide, even after injury
o Skeletal muscle is regenerated and grows by replication of satellite cells
 Constitute a reserve pool of stem cells that can become myocytes after injury
 Delta-like ligands (Dll) triggers Noth signaling which stimulate proliferation of
satellite cells
o Heart may have progenitor like cells that can generate progeny only after injury
 Cornea
o Corneal epithelium maintained by limbal stem cells (LSCs)

14.29

Major growth factors involved in tissue regeneration and healing (TGF-B, EGF, PDGF, FGF

Healing process is separated into regeneration and repair

 Regeneration: lost or damaged tissue is completely restored by cell proliferation

o Like the growth of an amputated limb in amphibians
o Tissues with high proliferative capacity can regenerate after injury (skin and GI epithelia,
hematopoietic system, liver)
 Repair: restores some original structures, can cause structural derangements(scar formation)
Growth Factors: drive cell proliferation (control cycle entry and progression), funx as ligands, bind
receptors and deliver signals to target cells, signals stimulate transctiption of genes

 EGF
o Source: platelets, macrophages, saliva, urine, milk, plasma
o Produced in healing wounds in keratinocytes, macrophages, and inflammatory cells
o Mitogenic for keratinocytes, fibroblasts, hepatocytes
o Stimulates keratinocute migration
o Stimulates granulation tissue formation
 PDGF
o Source: platelets, macrophages, endothelial cellsm keratinocytes, smooth muscle cells
o Has 2 chains, three isoforms
o Stored in platelet granules and released on plateler activation
o Causes migration of PMNs, macrophages, fibroblasts, and smooth muscle cells to area of
inflammation and healing skin wounds
o Activiates macrophages, PMNS, and fibroblasts
o Mitogenic for fibroblasts endothelial cells, and smooth muscle cells
o Stimulates production of MMPs, fibronectin, and HA
o Stimulates angiogenesis and wound contraction (PDGF B and C- also stimulate activation of
hepatic stellate cells)
 FGF
o Source: macrophages, mast cells, T lymphocytes, endothelial cells, fibroblasts
 o uses 4 tyrosine kinase receptors
o FGF-7= keratinocyte growth factor (KGF)
o FGF-2 and KGF contribute to re-epithelialization of skin wounds
o Promotes migration(chemotactic) of fibroblasts and keratinocytes
o mitogenic for fibroblasts and keratinocytes
o stimulates angiogenesis (FGF-2), wound contraction and matrix deposition
 TGF-B
o Source: platelets, T lymphocytes, macrophages, endothelial cells, keratinocytes, smooth
muscle cells, fibroblasts
o When active, binds Type I and II cell surface receptors with serine/threonine kinase activity
and triggers phosphorylation of Smad 4 which form heterodimers, enters nucleus,
associates with other DNA BPs, and activates or inhibits gene transcription
o Has multiple/often opposing effects depending on the tissue and injury type (pleiotrophic)
o Chemotactic for PMNs, macrophages, lymphocytes, fibroblasts, and smooth muscle cells
o Stimulates TIMP synthesis, angiogenesis, and fibroplasias
 Enhances production of collagen, fibronectin, and proteoglycans
o Inhibits production of MMPs and keratinocyte proliferation
o Growht inhibitor for epithelial cells (blocks cell cycle by increasing INK4/ARF and Cip/Kip)
o Inhibits collagen degradation by decreasing matrix proteases and increasing protease
inhibitors
o Involved in development of fibrosis and chronic inflammatory conditions
o High TGF-B occurs in hypertrophic scars
o has a strong anti-inflammatory response but may enhance some immune funxs

14.30

Events in Cell growth and proliferation (related to regeneration and healing- liver as example)
 LIVER

 lobes that were resected DO NOT regrow, but the ones still there enlarge (compensatory
growth/hypersplasia)
 proliferation triffered by combones action of cytokines and GFs
 except TGF-a, hepatocyte replication is dependent on paracrine effects of GFs and cytokines
(HGF and IL-6)
 2 major restriction points: G0/G1 and G1/S
 Proto-oncogenes c-FOS and c-JUN are activated, dimerize and form AP-1, c-MYC, and NF-kB,
STAT-3, and C/EBP
 Quiesent hepatocytes become competent to enter cell cycle through priming phase, mediated
by cytokines TNF and IL-6 and components of complement system
 Under HGR, TGF-a, and HB-EGF stimulation, primed hepatocytes enter cell cycle and undergo
replication
 NEPI, Serotonin, Insulin, thyroid and growth hormone act as adjuvants for liver regeneration
(facilitate entry of cells into cycle)

14. 31

How collagen contributes to wound healing. Compare Type I and II with IV and VII.

Collagen: most important common protein in world, provides extracellular framework for all
multicellular organisms, without it we would be a blob, composed of 3 chains that form a trimer in
shape of triple helix, Gly-X-Y, has 2 specialized amino acids (4-hydroxyproline, hydroxylysine)

Types I (ubiquitous in hard and soft tissues),II (in cartilage, intervertebral disk, vitreous), III, V, XI are
fibrillar collagens : triple helical domain is uninterrupted for >1000 residues
Type IV collagens (in basement membrane) have long but interrupted triple helical domains and form
sheets not fibrils, main cmpnts of basement membrane (with laminin)

Type VII (in anchoring fibrils ar dermal-epidermal junxs) forms anchoring fibrils between some epithelial
and mesenchymal structures (epidermis and dermis)

14. 32

Roles of ECM components in repair and healing

 Essential for wound healing bc they provide the framework for cell migration, maintain correct
polarity for re-assembly of multilayer struxs, and participate in angiogenesis
 Regulates growth, proliferation, movement, and differentiation of the cells living with in
 In it are fibroblasts, macrophages, others produce GFs, cytokines, and chemokines that are critical
for regeneration and repair
 Provides the scaffolding for tissue renewal- normal strux can only be reinstituted if the ECM is not
damages, if it is there is collagen deposition and scar formation
 Composed of:
o Fibrous structural proteins
 Collagen
 Elastins
 Make up central core of elastic fibers
 Surrounded by network of microfibrils- made of fibrillin
o Form scaffold for elastin
o Influence availability of active TGFB in the ECM
 Lots in large blood vessels, uterus, skin, and ligaments
o Adhesive glycoproteins
 Connect matrix elements to one another and to cells
 CAM 4 families:
 IG family CAMS
 Cadherins: with integrins links cell surface with cytoskeleton by binding
actin and intermediate filaments, forms zonula adherins and desmosome
junxs (migration of keratinocytes in re-epithelialization of skin wounds is
dependent on formation of desmosomes), linkage with cytoskeleton occurs
through catenins
 Integrins: bind ECM proteins like fibronectin, laminin, osteoponin providing
connection between cells and ECM and bind adhesive proteins in other cells
establishing cell-to-cell contact
o Fibronectin: protein, binds collagen, fibrin, proteoglycans, cell
surface receptors, tissue and plasma form, plasma form binds fibrin
which helps stabilize blood clot that fills gap created by wound and
serves as substratum for ECM deposition and formation of
provisional matrix in wound healing
o Laminin: most abundant glycoprotein in basement membrane, form
network with collagen type IV ,mediates attachment of cells to CT
substrates
 Selectins: leukocyte/endothelial interactions
 Transmembrane receptors
  Other adhesive proteins:
 SPARC/osteonectin- tissue remodeling in response to injury and
angiogenesis inhibitor
 Thrombospondins- inhibit angiogenesis
 Osteoporin (OPN)- regulated calcification, mediator o leukocyte migration
involved in inflammation, vascular remodeling, and fibrosis
 Tanascin- morphogenesis and cell adhesion
o Proteoglycans and hyaluronan
 Provide resilience and lubrication
 GAGS form proteoglycans- organize ECM, regulate CT strux and permeability
 4 GAG families
 Heparin sulfate-
 Chondroitin sulfate
 Keratin sulfate
 Hyaluronan (HA)- produced in plasma membrane (all others in golgi), lots of
repeats, binds lots of water, gives CT the ability to resist compression forces,
helps provide resilience and lubrication to CT (Esp in joints), increases in
inflammatory diseases, LMW (low molecular weight) HA recruits leukocytes
to inflamed sites, stimulated production of inflammatory cytokines and
chemokines
 These assemble to form:
o Interstitial matrix
 Between epithelial, endothelial, smooth muscle cells, CT
 Consists of fibrillar, nonfobrilliar collagen, elastin, fibronectin, proteoglycans and
hyaluronan
o Basement membrane
 Closely associated with cell surfaces
 Consists of nonfibrillar collagen (mainly IV), laminin, heparin sulfate, and
Proteoglycans
 If injury is too severe or chronic, damage to parenchymal and stromal framework is done, and
healing cannot be accomplished by regeneration, instead repair is done by deposition of collagen
and ECM components which cause the formation of a scar
o A fibroproliferative response
o Patches rather than restores tissue
o Features:
 Inflammation
 Angiogenesis (EXTREMELY important in wound healing)
 Migration and proliferation of fibroblasts
 Scare formation
 CT remodeling
o If damage persists, inflammation becomes chronic, leading to excess deposition of CT=
fibrosis
o In most healing a combo of regeneration and repair occurs
 Distribution influenced by:
 Proliferative capacity of cells of tissue
 Integrity of ECM
 Resolution or chronicity of injury and inflammation
14.33

How angiogenesis contributes to tissue repair with wound healing. Control of this by Notch signaling.

Angiogenesis: involves branching and extension of pre-existing cessels and recruitment of endothelial
progenitor cells (EPCs) from bone marrow

From pre-existing vessels:

 Vasodilation in response to NO, VEGF induced increased permeability of vessel

 Proteolytic degradation of basement membrane of parent vessel by matrix metalloproteinases
(MMPs) and disruption of cell-to-cell contact by plasminogen activator
 Migration of endothelial cells toward stimulus
 Proliferation of endothelial cells
 Maturation
 Recruitment of periendothelial cells to form mature vessels

From EPCs

 EPCs recruited from bone marrow into tissues to initiate angiogenesis

 Cells express markers of HSCs and VEGFR-2 and VE-cadherin

Notch Pathway: modulates angiogenesis

 Promotes proper branching of new vessels and prevents excessive angiogenesis by decreasing
responsiveness to VEGF
 Ligands and receptors are membrane-bound and conserved
 5 ligands in mammals (Jagged 1,2, Dll 1,3,) and 4 receptors (Notch1,2,3,4)
o Receptors have EGF-like repeats
 Dll4 only on arteries and capillaries
 Leading cell/tip cell undergoes proliferation dn migration but stalk cells maintain connections with
vessel
o VEGF indices Dll4 in tip cells
o Notch 1 and 3 are expressed in stalk cells
 Interaction of Dll4 and Notch receptors leads to 2 step Proteolytic cleavage of the receptor,
releasing Notch intracellular domain, which translocates to nucleus and activates genes that
dampen responsiveness to VEGF
 Blocking Dll4 causes increased proliferation of endothelial cells and capillary sprouting
 Blocking VEGF has the opposite effect
14.34

VEGF and its receptors in angiogenesis

VEGF is the most important GF in adult tissues undergoing angiogenesis

Its secreted by mesenchymal and stromal cells

It induces the migration of EPCs and enhances proliferation and differentiation of these cells at sites of
angiogenesis (can also be stimulated by FGF-2)

Table 3-3. Vascular Endothelial Growth Factor (VEGF)

 Proteins Family members: VEGF (VEGF-A), VEGF-B, VEGF-C, VEGF-D
  Dimeric glycoprotein with multiple isoforms
  Targeted mutations in VEGF result in defective vasculogenesis and angiogenesis.
Production Expressed at low levels in a variety of adult tissues and at higher levels in a few sites, such as
podocytes in the glomerulus and cardiac myocytes
Inducing Hypoxia
agents
  TGF-β
  PDGF
  TGF-α
Receptors VEGFR-1
  VEGFR-2 (a tyrosine kinase receptor- THE MOST IMPORTANT IN ANGIOGENESIS)
  VEGFR-3 (lymphatic endothelial cells)
  Targeted mutations in the receptors result in lack of vasculogenesis
Functions Promotes angiogenesis
  Increases vascular permeability
  Stimulates endothelial cell migration
  Stimulates endothelial cell proliferation
  VEGF-C selectively induces hyperplasia of lymphatic vasculature
  Up-regulates endothelial expression of plasminogen activator, plasminogen activator inhibitor 1, and
collagenase

14.35

Inflammation, proliferation, maturation steps in wound healing. Role of granulation tissue.

3 Wound healing phases: (they overlap)

 Inflammation: from platelet adhesion and aggregation into a clot

 Proliferation: formation of granulation tissue, proliferation and migration of CT cells and re-
epithelialization of wound surface
 Maturation: ECM deposition, tissue remodeling, wound contraction

Steps in Healing

 Formation of blood clot by activation of coagulation pathways

 o Clot has red cells, fibrin, fibronectin, complement components; stops bleeding and makes
scaffold for migrating cells (attracted by GFs, cytokines, chemokines)
o Clot larger if wound larger
o Within 24hrs neutrophils appear at margins- release Proteolytic enzymes that clean wound
 Formation of granulation tissue
o Fibroblast and endothelial cells proliferate in first 24-72 hrs to form granulation tissue:
hallmark of tissue repair
 Angiogenesis and fibroblast proliferation
 New vessels are leaky, plasma proteins and flud lead, so edematous
 Amount formed depends on deficit and intensity of inflammation
 Cell proliferation and collagen deposition
o Neutrophils replaced by macrophages by 48-96 hrs
o Key cell components of tissue repair; clean debris, fibrin, and foreign material
o Fibroblasts migrate
o Collagen fibers present at margins (at first vertically oriented and do not bridge)
o In 24-48 hrs, spurs of epithelial cells moce from wound edge along cut margins of dermis,
depositing basement membrane components as they move
o They fuse in midline to produce thin epithelial layer that closes the wound (slower in
secondary)
o Macrophages stimulate fibroblasts to produce FGF-7 and IL-6 which enhance keratincyte
migration and proliferation
o Other mediators pr reepithelialization=HGF and HB-EGF, CXCR3
o Collagen fibrils become more abundant and begin to bridge incision
 Type III, fibrin, fibronectin at first, replaced by type I collagen
 TGFB is most important fibrogenic agent
 Scar formation
o Leukocytic infiltrate, edema, increased vascularity disappear in 2 nd week
o By end month 1, scar is mostly acellular CT without inflammatory infiltrate and covered by
epidermis
 Wound contraction
o Generally in large surface wounds (so important in secondary)
o Helps close wound by decreasing gap between dermal ridges and by reducing SA
o Initial steps: formation of myofibroblasts at edge of wound- smooth a-actinin and vimentin
 Connective Tissue remodeling
o Replacement of granulation tissue with scar involves changes in ECM
o Balance between synthesis and degradation of ECM
 MMPS degrade ECM and collagen
o Collagenases and inhibitors are essential in remodeling of ECM
 Recovery of Tensile Strength
14.36

Wound healing by primary and secondary intention. Wound Contraction.

Primary union/first intention: simplest repair by a clean, uninfected surgical incision approcimated by
surgical sutures; thin scar

Secondary: more complex; wound creates large defect in skin surface and causes loss of cells and tissue;
healing involves more intense inflammatory reaction; formation of abundant granulation tissue;
extensive collagen deposition; substantial scar; contraction

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GFs known to participate in healing (TNF, TGF, VEGF, EGF, PDGF, FGF)

TNF, PDGF, TGF-B, FGF promote migration of fibroblasts to site of injury

Subsequent proliferation promoted by PDGFm EGF, TGF-B, FGF, and IL-1, TNF (mainly from
macrophages, also from inflammatory cells and platelets)
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What contributes to wound strength/How quickly achieved. Why 5 small incisions for laparoscopy are
better than 1 big for laparotomy.

Wound Strength

 Fibrillar (mostly type I) for major part of CT in repair sires and are essential fo strength in healing
wounds
 Net collagen accumulation depends on increased collagen synthesis and decreases degradation
 At end of wk 1 after surgical wound, strength is 10% of unwounded skin
 Strength increases rapidly over next 4 weeks, slows at third month following incident, plateau at 70-
80% tensile strength of unwounded
 Lower tensile strength may persist for life
 Recovery of strength is due to excess of collagen synthesis over collagen degradation, later from
structural modifications of collagen fibers (cross-linking, increased fiber size) after synthesis stops

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MMPs in wound remodeling

MMPS degrade ECM and collagen

o Interstitial collagenases (MMP1,2,3) cleave Type I, II, III

o Gelatinases (MMP 2, 9) cleave amorphous collagen and fibronectin
o Stromelysins (MMP 3,10,11) cleave proteoglycans, laminin, fibronectin,
amorphousncollagens
o ADAMS
 17 cleaves precursor TNF and TGF-a, releasing active molecules
 Involved in bronchial asthma and microangiopathies
 Produced by fibroblasts, macrophages, neutrophils, synovial cells, epithelial cells, PDGF, FGF, IL-1,
TNF, macrophages; inhib by TGF-B, steroids
 Have 180 residue zinc-protease domain in common
 Collagenases cleaves collagen
 Collagenases inhibited by inhibitors of metalloproteinases

Collagenases and inhibitors are essentioal in remodeling of ECM

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Pre and post-operative factors that modify wound healing and outcome (wound dehiscence)

Systemic Factors:

 Nutrition: protein and vit C deficiency inhibit collagen synthesis and retard healing
 Metabolic status: Diabetes delays wound healing bc of microangiopathy
 Circulatory status: inadewuate blood supply (varicose veins) retards venous drainage->impaired
healing
 Hormones (glucocorticoids
 0: anti-inflammatory effect, inhibit collagen synthesis

Local Factors:

 Infection: single most important cause of delay in healing, results in persistent tissue injury and
inflammation
 Mechanical factors: delay healing by compressing blood vessels or separating edges of wound
 Foreign bodies: impediments to healing
 Size, location, and type f wound: if in richly vascular area-heal faster than in poorly vascularized;
small ones heal faster and scar less than large ones

Wound Dehisence

 From inadequate formation of granulation tissue or scar formation (can also form an ulcer if
inadequate vasculatization)
 Most common in adbomnal surgery from increased abdominal pressue
o Vomiting, coughing, ileis generates mechanical stress

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Complications of wound healing: Hypertrophic scar, keloid, excessive granulation tissue, contracture

Hypertrophic scar

 From accumulation of excessive amounts of collagen

 Generally develop after thermal or traumatic injury and involve deep layers of dermis
 Collagen persists through TGF-B and establishes focal adhesions

Keloid

 if scar tissue grows beyond boundaries of the original wound and does not regress
 individual predisposition
 more common in African Americans- don’t know why

Excessive granulation tissue

 protrudes above level of surrounding skin and blocks re-epithelialization

 must be removed be cautery or surgical excision to permit restoration of continuity of the
epithelium
 may recur (between benign and malignant)

Contracture
 contraction is an important part of normal healing but if exaggerated, causes contracture and
deformities
 prone to develop on palms, soles, anterior aspect of thorax
 common after serious burns
 can compromise joint movement

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Fibrosis/scarring from chronic inflammation

Fibrosis: excessive deposition of collagen and ECM components in a tissue, often in chronic disease,
mechanism similar to mechanism of skin wound healing, just persistence of injurious stimuli which leads
to organ dysfunction/failure, injury persistence leads to chronic inflammation which activates and
proliferates macrophages and lymphocytes, which increases cytokine production and fibrogenic GFs

Host response:

 classically activated macrophages ingest microbe

o cytokines that activate classical macrophages are produced by TH1 cells (IFN-y and TNF)
 alternatively ingested macrophages suppress microbicidal activities and remodel tissues and
promote angiogenesis and scar formation
o Alternative activation is induced by IL4 and IL13 from TH2 cells, mast cells, and eosinophils,
o Alternative produce TGF-B and other GFs in repair process
 Triggers of activation of TGF-B may be ROS, necrosis or apoptosis
 Myofibroblasts are main collagen sources in lung and kidney fibrosis
 Stellate cells are in liver cirrhosis
 Role of OPN: in fibrosis of heart, lung, kidney, liver; blockage decrease formation of granulation and
scarring; mediator of differentiation of myofibroblasts; induced by TGF-B
 Fetal cutaneous wounds heal without scar formation
o Possibly from TGF-B secretion, lack of OPN, absence of TH2