Wound Healing

Contributor: Falko Krause
Supervision: Michael Schumann

Introduction

General Phases of Wound Healing

Wound healing can be differentiated into three major phases (as seen in Fig. 1):
  • Inflammatory phase (1)

    The wound is rinsed by blood and filled with blood-collagen and fibrin constituents.

  • Proliferative phase (2)

    Resorption of blood-collagen and reepithelialization. The reepithelialization is a movement of the surrounding epithelial cells to close the wound.

  • Maturation and remodeling phase (3)

    Growth of new epithelial cells.

This is just a basic overview for a more detailed overview you can visit this site. This webscript will discuss the reepithelialization in the proliferative phase. To get a deeper understanding of the events that lead to the reepithelialization some basic knowledge about cell movement has to be understood. This will be discussed in the following section.


Figure 1: schematic drawing of major epithelial wound healing phases (Source:??)

Cell Movement


Figure 2: cell movement overview (Source: Reference [2])		 Cell movement overview (Fig. 2)
The fist step in the cell movement is an extension of the cell membrane (1). The extensions are called lamellipodia. After a certain amount of extension the lamellipodia attaches itself to the surrounding substratum by focal adhesion (2). This is followed by a forward flow of the cytosol (3) and the retraction of the rear of the cell (4) which also means a detachment from the surrounding substratum (5).

Lamellipodia extension (Fig. 3)
In more detail the lamellipodia extension is characterized by the polymerization of action filaments lead by profilin in the leading membrane(1). The actin filaments are attached to the membrane by myosin I. To stabilize the extension actin filaments are cross-linked into networks and bundles(2). At the same time cofilin induces a degradation of the actin filaments at the end that is opposed to the direction of the movement of the cell(3).


Figure 3: lamellipodia extension (Source: Reference [2])

Recent Model


Figure 4: recent model of wound healing signal pathway (Source: Reference [2])

In the recent model of wound healing the three small GTPases Rho, Rac and Cdc42 are activated through external signals. Each small GTPase is thought to trigger a different morphological remodeling in the cell. Cdc42 is thought to trigger the formation of Filopodia (small lamellipodia like extensions), Rac is supposed to trigger the lamellipodia formation with the help of cofilin as described earlier and Rho triggers the stress fiber assembly in the moving cell as well as the focal adhesion. The small GTPases are supposed to be activated in the chronological order they were described before which also can be seen in Fig. 4.

 

Drosophila Melanogaster Dorsal Closure


Figure 5: Drosophila melanogaster embryonic development (Source: here )		 During the early development of Drosophila melanogaster embryos a morphological change occurs that is called dorsal closure. The dorsal closure occurs during the stages 12 through 15 (of 17 stages where the 18th stage would be the hatching). This closure has similar properties as a healing wound since there is no more cell division taking place in the involved region at that stage of the development. Using Drosophila melanogaster as a model for wound healing is advantagous not only because the researcher looks at preformed wound edges but also by the fact that knowledge of the genetics of Drosophila development is far advanced and may help to explain some of the newly described phenomena.

Furthermore its use as a model for wound healing is given by the fact that the genetics of Drosophila development are well researched as well as the bilayered structure of the closing membrane which simplifies observations.
Figure 6: Drosophila melanogaster dorsal closure (Source: here )

Wound Healing and Morphogenesis

Wound healing recapitulates morphogenesis in Drosophila embryos.

In the following sections the experimental approaches as well as their results described in Ref. [4] will be discussed. The approaches are based on previous research (Ref. [1], Ref. [3]) about the dorsal closure in Drosophila melanogaster. In particular the functions of the small GTPases Cdc42, Rho Rac are addressed as well as their chronological activation in a healing wound.

In the experiments GFP-Spaghetti-squash (GFP-sqh) transgenetic Drosophila mutants were used to produce stems where one of the three small GTPases is knocked out. Through GFP marking the actin in the cells was visualized.

Previous experiments exposed two major morphgenic changes during wound healing. The formation of actin cable on the leading edges of the cells surrounding the wound as well as the formation of filopodia. The actin cable was observed to act like a purse string drawing cell close to the center of the wound. Fig. 7 shows a visualization of both changes.


Figure 7: What is the chronological order of actin cable formation in leading edges (arrows) and filopodia formation (triangles)? What are the signals that trigger each morphological change?(Source: Reference [1])

Experimental Setup


Figure 8: time a time lapse confocal laser microscopy setup (Source: here )

Figure 9: dye laser		 To visualize the wound healing over time a time lapse confocal laser microscopy (Fig. 8) was applied. This means that in vivo cells were wounded and the healing process was visualized.

The wounds were created by laser ablation (Fig. 9) and mechanical (microinjection pipett) In Fig. 10 a-d selected cells were tinged to visualize the movement of cells during the wound healing. In Fig. 10 e-f the final closing process of the dorsal closure is visualized again with tinged cells. In both process no new cells emerged.
Figure 10: a-d tinged cells move into a wound e-f zipping phase of dorsal closure (Source: Reference [4])

Wound Types

Two types of wounds were created in the experiments:

Rho1 Mutants

The knock-out of the Rho1 gene in GFP-sqh Drosophila yielded the following observations:


Figure 11: a - wound closure in wild type GFP-sqh Drosophila; b - wound closure in Rho1 mutants takes twice as log as in wild type; plot - wound size over time in wild type (red) and Rho1 mutants (blue) (Source: Reference [4])
Figure 12: formation of local zipping fronts in Rho1 mutants (Source: Reference [4])

Cdc42 Mutants

The knock-out of the Cdc42 gene in GFP-sqh Drosophila yielded the following observations:
  • The actin cable in leading edge cells forms.
  • There is no filopodia or lamellipodia formation.
  • After 30 minutes the wound almost closed however there is an inability to seal the wound completely

These observations suggest a model of reepithelialization where Cdc42 acts as the signal for the formation of filopodia that draw neighboring cells close to each other. The local zipping fronts only act on short distances and result in a formation of weak adhesion over retracting filopodia. In a final step the weak adhesions are replaced by strong adhesions (Fig. 14).
The initial wound closure is lead by the formation of an actin cable in the cells at the edges of the wound. The formation of the actin cable is triggered by Rho1 and acts like a purse string shortening the time of wound closure.

The knock-out of the Rac gene did not effect wound healing.
Figure 13: wound closure of wild type (a) and Cdc42 mutants (b) (Source: Reference [4])

Figure 14: proposed model for epithelial fusion: i) filopodia from opposing cells attach and form weak adhesions ii) filopodia shortens and draw opposing cells close to each other iii) opposing cells form strong adhesions; blue - actin; yellow; weak adhesion molecules; orange - strong adhesion molecules (Source: Reference [3])

Discussion

Validation Methods

To validate the results found by the time lapse confocal laser microscopy experiments a set of alternative methods were used:
  • Phalloidin staining - Phalloidin is a toxin from the Death Cap mushroom that binds actin.
  • Transmission electron microscopy
  • Antibody staining - Antibodies were used to control if the knock-out strains did not produce the knocked out protein

These methods can not be visualized over time since they acquire an immobilization of the epithel.

Additionally different types of transgenetic Drosophila strains were used in the experiments.


Figure 14: a - phalloidin staining; b - sqh-GFP; c - α-catenin-GFP; d - GFP-actin; e&f - transmission electron microscopy (Source: Reference [4])

Summary


Figure 15: model of reepithelialization: cellular messengers function and chronological influence (Source: original content)		 The chronological order of the influence of the small GTPases Rho and Cdc42 are not as the recent model suggested. The presented experiments rather suggest that the initial wound closure is done by a purse string like mechanism (Rho) which is followed by a zipping together over smaller distances that end in a strong adhesion of the cells (Cdc42). Also the small GTPase Rac did not prove to have an effect in wound healing.

Outlook

There are still a lot of questions that have to be answered. It is still unclear what extracellular signals lead to the filopodia formation. What other extracellular signals play a role in wound healing?

Furthermore studies in vertebrate embryos should reveal to what degree the processes of wound healing are similar to those in Drosophila melanogaster.

Finally this research has the potential to modify therapeutic strategies for enhancing wound healing in the future.

References

[1] A. Jacinto, A. Martinez-Arias, and P. Martin. Mechanisms of epithelial
fusion and repair. Nat Cell Biol, 3(5):E117–E123, May 2001.
[2] Arnold; Zipursky S. Lawrence; Matsudaira Paul; Baltimore David; Darnell
James E. Lodish, Harvey; Berk. Molecular Cell Biology 4th ed.Chapter 18.6. Cell
Locomotion New York: W. H. Freeman & Co., 1999.
[3] Paul Martin and William Wood. Epithelial fusions in the embryo. Curr
Opin Cell Biol, 14(5):569–574, Oct 2002.
[4] William Wood, Antonio Jacinto, Richard Grose, Sarah Woolner, Jonathan
Gale, Clive Wilson, and Paul Martin. Wound healing recapitulates morphogenesis
in Drosophila embryos. Nat Cell Biol, 4(11):907–912, Nov 2002.