Connective Tissue

Livingston Van De Water, Ph.D.

Room MS-450; Phone: 2-9945
vandewl@mail.amc.edu
I. Connective Tissues: Definition and Cellular components
October 13, 2015, 10 am
II. Connective Tissues: Collagen, ECM synthesis
October 15, 2015, 10 am
III. Repair of connective tissues - October 16, 2015, 10 am
IV. Conference (Dr. Whyte, Pediatric Surgeon)
October 19, 2015, 10 am
V. Review October 20, 2015, 1pm

Learning Objectives-Lecture I
Connective Tissues: Definition and Cellular
components

What is connective tissue?

What does it do?

What are the prominent cell types?

What are the functions of these cells?

Connective tissue of skin (dermis)

Basement membrane
(or basal lamina):

Epidermis is
an epithelium

Dermis is
connective
tissue

Connective tissue:
(fibroblasts, mast cells
macrophages,
lymphocytes)
Connective tissue also
termed stroma, or
interstitial matrix
3
Alberts et al. MBOC. 4th ed. Garland. 2002

Fibrosis: a Disease of Connective Tissue

Deep thermal or
traumatic injury

Excessive inflammation,
exuberant wound healing

Hypertrophic scar
4

Hypertrophic scar histology

Epithelial tissue (epidermis)

Epidermis

Normal
dermis
Connective tissue (dermis)

Hemotoxylin & Eosin (H&E)

Hypertrophic scar nodule

Trichrome stain
5

What is connective tissue?

Provides support and connecting framework for all tissues of the body. Comprised
of cells and extracellular matrix (ECM). Cells generally are well-separated by ECM
components. Connective tissue supplied by blood, lymphatic vessels and nerves.
By contrast, epithelial cells are not separated by interstitial collagens and do not
have blood or lymphatic vessels.

Major cell types:

Fibroblasts
Macrophages
Mast cells
Lymphocytes

Kumar et al. Robbins & Cotran Path. Basis of Disease

Fig 3-14. 2005.

Interstitial matrix? ECM components in connective6

tissues not in basement membranes.

What Do Connective Tissues Do?

Support blood vessels and lymphatic vessels
Store metabolites, hormones and provide nutritional
support to epithelium
Provide mechanical protection of organs and a structural
framework for organ function.
Provide battleground for host response to infection and
injury

Blood vessels are supported by the

dermis, but are not connective tissue

Vascularization of:
Papillary dermis
Sebaceous gland
Sweat gland
Hair follicle

Artery
Vein
Habif TP, Clinical Dermatol, 1990.

Nutritional & structural support for

epithelium
Epidermis (epithelium)

Store metabolites, hormones

and provide nutritional
support to epithelium
Basement membrane,
contributed in part by
connective tissue
Blood vessel
Fibroblasts
Extracellular
matrix

Dermis (connective tissue)

Will discuss more about

mechanical features
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Battleground following infection and injury

Innate immunity (750 million yrs

old):

Adaptive (verts:350-500 million yrs old):

Immediate. Prior exposure to

microbe not required

Follows innate immunity. Requires cell

recruitment.

No memory and limited specificity.

Reacts with ~1000 microbe
products. PAMPS and DAMPS!

Long lasting memory, great specificity.

Reacts with millions of microbial antigens
Abbas et al. Cell. and Molec. Immunology, Eighth ed., 2015.

10

Major Cell Components of

Connective Tissue
Lymphocytes: T cells (mediators of cellular immunity),
Natural killer (NK) cells (innate immunity) and B cells
(secrete immunoglobulins).
Macrophages: are phagocytic cells that secrete proteases,
present antigens to lymphocytes, produce cytokines (e.g.,
Il-1). Centrally important in tissue repair and regeneration
Mast cells: release vasoactive mediators (e.g., histamine)
and chemotactic factors.
Fibroblasts: Secrete and deposit interstitial connective
tissue components (including proteoglycans, collagens,
elastin and non-collagenous proteins), secrete proteases
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and generate mechanical force.

Connective tissue lymphocytes

B cell

T cell

NK cells

Each of these lymphocyte classes can

be found in connective tissues. In
resting CT their numbers are lower
than during an inflammatory reaction,
tissue injury or infection.
Plasma cells (a version of B cells) are
prominent constituents of loose
connective tissues (e.g., GI and
respiratory tracts). These produce large
amounts of a single antibody molecule
12

Abbas et al. Cell. and Molec. Immunology, Eighth ed., 2015.

B lymphocyte (plasma cell)

Key points:

B cells are the source of all

antibodies

These antibodies are critical

components of a number of
important defense mechanisms.

These effector functions are

performed in collaboration with
specific cell types, many also
residents of connective tissues

Plasma cell: Note the prominent

ER and Golgi apparatus suggesting
a highly active secretory cell:13
Abbas et al. Cell. and Molec. Immunology, Eighth ed., 2015.

Connective tissue
macrophages and their origins
Long-lived resident
macrophages:
Derived from
precursors in the
fetal liver in
humans

Adult, recruited
macrophages:
Monocytes arise
from bone marrow
precursors and
mature monocytes
are blood borne.
Abbas et al. Cell. and Molec. Immunology, Eighth ed., 2015.

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Macrophages: origins & travel

Macrophages are components of the
mononuclear phagocyte system.
Tissue macrophages are in connective tissue,
liver, spleen, lymph nodes and lungs (alveolar
macrophages).

Monocytes migrate into various tissues and

differentiate into macrophages. Monocyte halflife (1 day); tissue macrophages (mo->yrs).

Extravasation (pass out of vessel) mediated by

adhesion molecules and chemotactic factors
(chemicals promoting migration).
Maturation from monocyte to macrophage
occurs in extravascular space.
Activation occurs during inflammation.
Kumar et al. Robbins & Cotran Path. Basis of Disease. Figs 2-6, 2-27. 2005.

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Macrophages: phagocytosis
Opsonins: host
proteins that coat
particle, making it
palatable to cells.

FUSION

Macrophages once activated become phagocytic cells (sometimes termed

professional phagocytes):
Recognition: opsonized, injurious agents via cell surface receptors
Engulfment and fusion with lysosome: complex process requiring membrane
remodeling, cytoskeleton and intracellular signaling.
Killing and degradation: via oxygen-dependent (reactive oxygen species) and
lysozomal enzymes.
Kumar et al. Robbins & Cotran Path. Basis of Disease. Figs 2-11. 2005.

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Phagocytic function in the skin

Mosbys Color Atlas of Dermatol, 1998.

Macrophages ingest
tattoo pigments.

17

Macrophages and dendritic cells

present antigen to lymphocytes

18
Abbas et al. Cell. and Molec. Immunology, Eighth ed., 2015.

Macrophages and dendritic cells

present antigen to lymphocytes

Macrophages phagocytose microbes and proteins degrade them (in

lysozomes) and present peptides to T lymphocytes.
Activated T-cells proliferate and can secrete cytokines that also
activate macrophages
19
Abbas et al. Cell. and Molec. Immunology, Eighth ed., 2015.

Macrophages and dendritic cells

present antigen to lymphocytes
Activated T-cells effector cells
secrete cytokines that both activate
the APC to amplify the response
Effector T-cells

and expand the T-cell effector

population.

Kumar et al. Robbins & Cotran Path. Basis of Disease. Figs 6-2. 2005.

In viral diseases these effector T-cells

lyse the virally infected, host cell.
Also activated APCs and
macrophages destroy the ingested
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microbes.

Roles of macrophages in
inflammation and repair
M1

M2

Macrophage
characteristics
support their
classification into two
groups: M1 and M2
with contrasting
functions.
Note that the
regulation of these
macrophage subtypes
is governed by
cytokines (secreted
factors that regulate
cells of the innate and
adaptive immunity)
21

Abbas et al. Cell. and Molec. Immunology, Eighth ed., 2015.

Macrophages as secretory cells

Growth factors: proteins which
promote the growth and
activation of other cells.
Example: TGF-beta activates
fibroblasts.
Some growth factors (VEGF,
FGF) are angiogenic.
Cytokines: factors which
influence other cells. Example:
TNF, IL-1. Cytokines can affect
more than one cell type/function
(pleiotropic). Functions
overlap with other cytokines
(redundant).

Kumar et al. Robbins & Cotran Path. Basis of Disease. 2005.

Metalloproteinases (MMPs) &

Collagenases: enzymes which
degrade extracellular matrix
proteins, including collagen
22 and
elastin.

Macrophages as secretory cells

Macrophages are elements in a network of inflammatory signals:

cytokines secreted by macrophages and lymphocytes (and other
connective tissue cells) direct the proliferation and activation of other
cells.
Kumar et al. Robbins & Cotran Path. Basis of Disease. 2005.

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Mast cells

Degranulation can be effected by: physical trauma,

immune reactions (antigen with IgE), complement
proteins, leukocyte mediators, neuropeptides and
cytokines

Mast cells surround blood

vessels in connective tissue.

Mast cells
(dark)
Fat cells (red)

Metachromatic granules present in

cytoplasm are rich in glycosaminoglycans.
Metachromasia= color different than the
applied stain.

Histamine causes
dilation of arterioles
and increases
permeability of
venules

Mast cell histamine in granules

is released by degranulation.

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Kumar et al. Robbins & Cotran Path. Basis of Disease. 2005.

Mast cell effector functions

Biogenic amines
and enzymes
are stored
preformed in
granules ready
for immediate
release.
Cytokines and
lipid mediators
are synthesized
upon mast cell
activation and
promote
inflammation
and tissue
damage

Abbas et al. Cell. and Molec. Immunology, Eighth ed., 2015.

25

Mesenchymal stem cells and their lineages

A I Caplan and S P Bruder, 2001

26

Fibroblasts and related cells

Cell type:

Location:

Fibroblast

Widespread in
Deposit & remodel Col1
connective tissue & 3-rich matrix

Chondrocyte

cartilage

Deposit Col2-rich matrix

Osteoblast

bone

Deposit Col1 &

hydroxyapatite crystals

Adipocyte

fat

Lipid accumulation &

coalescence

Smooth muscle Numerous

cell
organs

Function:

Contractile function.
Elastin & Col 1
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Fibroblast secrete proteases involved

in tissue remodeling
Deposit and organize collagen-rich
extracellular matrix.
Provide functionally appropriate
contractile force (resting skin vs.
wounds).
Respond to mechanical signals
Degrade (remodel) via proteolysis.

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Kumar et al. Robbins & Cotran Path. Basis of Disease. 2005.

Fibroblasts assemble collagen

fibrils in skin
Fibroblast

Collagen fibrils are oriented

at right angles to each other

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Variations in Dense Connective Tissue Structure

Collagen bundles:
Irregularly arranged

Collagen bundles:
Regularly arranged

Dermis in skin, note

wavy pattern

Note the linear pattern

of fibroblasts in tendon

Dermis as visualized
by polarizing
microscopy
[Kierszenbaum: Histology and
30Cell
Biology, Mosby, 2002]

Structure of Interstitial Collagens

Collagen fibril
64 nanometer (nm) gap

Electron micrograph - note periodicity

which reveals staggered array of
collagen molecules
Collagen molecules are organized
into a quarter stagger array in
fibrils
Each collagen molecule is 300 nm
long - also termed tropocollagen
Each tropocollagen is triple (alpha)
helix often two from one gene (e.g.,
alpha 1) and one from another gene
(e.g., alpha 2)
Each molecule has the sequence GlyX-Y..X is frequently Pro and Y
Hydroxy-Pro
31

Uitto et al. Chapter 19 in Freedberg et al Dermatology in General Medicine (5th ed).

Major Types of Collagens

Fibrillar
collagens

Fibrilassociated
Network
forming

32

Fibroblast and contractile forces

Skin tone is, in part, because of ambient
contracile forces generated by fibroblasts. Skin
is stiffer along Langer's lines than across the
lines.
If surgeon cuts across Langer lines generates
larger scar than if cut within these lines.
Fibroblasts generate tension in tissues through
actin cytoskeleton and contact with extracellular
matrix at focal adhesions.
Fibroblasts also respond to mechanical signals
(probably also through focal adhesions)
activating intracellular signaling networks.

33

Collagen provides tensile strength

in connective tissues

Tensile strength

Triple helical
collagen is
rigid

34

Fibroblasts exert force on the

extracellular matrix
focal adhesions (green)

Outside cell
Cell membrane
Inside of cell
focal adhesions
(green in fig at left)

Actin containing
cytoskeleton
(red)

Fibroblasts elaborate focal adhesions:

Connect the extracellular matrix with the actin
cytoskeleton.
Serve as signaling centers within the cell. 35

Myofibroblast Differentiation and Function

Myofibroblasts are contractile

fibroblast
On silicon (wrinkling)

myofibroblast

Type I Collagen mRNA (in situ hybridization)

Key points:
Fibroblast differentiate into myofibroblasts
Differentiation involves signals from immune
cells, ECM and mechanical environment
Myofibroblast effector functions include
enhanced contraction and ECM production

Dermal fibroblasts
Tomasek et al (2002). Nature Reviews Molecular Cell
Biology.

Van De Water, Varney, Tomasek. Adv.

Wound Care (2013)

Wound
36
myofibroblasts

Myofibroblasts persist in wound

pathologies: scarring and organ fibrosis
Reversion

Apoptosis

Growth factors,
cytokines, mechanical,
extracellular matrix TGF-b autocrine loop

Persistent
differentiated state

37

Hypertrophic Scarring
Raised scars
Remain within boundary of wound
Can regress spontaneously (months-yrs)
Arise from injury to the deep dermis (burn,
traumatic injury, prolonged inflammation)
Defined as a fibroproliferative/fibrocontractive
disorder
Epidermis

Trichrome stain

Nodule

Polarizing
Polarizing microscopyNodule
microscopy

Van De Water, Varney, Tomasek. Adv. Wound Care (2013)

Myofibroblasts (SMC -actin

IH)
38

Summary
I. Connective Tissues: Definition and
Cellular components
What are functions of connective tissue?
What are the prominent cell types?
What are the functions for each of these
cells?

39

Questions
Your lab is studying connective tissue cells and obtains
a knock-out mouse that is unable to make
macrophages. For which of the following connective
tissue functions do you expect to see an effect:
A. Collagen synthesis.
B. Immunoglobulin production.
C. Histamine release.
D. Uptake of injected carbon particles.

40

Questions
Your skin tone is maintained by a combination of the
mechanical rigidity of the extracellular matrix and the
force exerted by fibroblasts on it. Which of the
following cell structures is directly responsible for the
generating the observed tension:
A. Cell membrane
B. Integrins
C. Stress fibers
D. Mitochondria
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Questions
Which collagen is found within basement
membranes but not connective tissues?
A. Type I
B. Type III
C. Type IV
D. Fibril associated collagens (e.g., Type IX).
E. Type V

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