Sunday, 4 March 2012

Xiaflex in cells - Trying to help you understand, maybe

So today I am going to try and review this paper In Vitro Study of Novel Collagenase (XIAFLEX®) on Dupuytren's Disease Fibroblasts Displays Unique Drug Related Properties. This paper is open access so you might like to take a look for yourself or have it open at the same time as you read this and it might help. I don't understand all of it so good luck!

I say try because there is a lot of scientific content in this paper, a lot of stuff that I have not done in the lab before and this makes it harder for me to work out exactly what it means, I will however try my best to understand it and where possible try and explain it in as simple terms as possible.

To start with for those who don't know Xiaflex is a treatment for Dupuytren's disease (DD) and potentially Ledderhose disease (LD), it is a collagenase and the main component of the lumps in DD and LD is collagen and so it breaks them down.

Right onto the paper, where possible I shall try and bullet point it and I am going to place a summary at the end where I will put what I consider to be the key points.

Introduction:

  • DD is a common and benign disorder. 
  • The cause(s) of DD and LD are unknown however DD has been linked to genetics, smoking, diabetes, alcohol, anti-epilepsy medication, occupation and local trauma (so all the usual stuff). 
  • The group of people at the most risk are Caucasian men of North European descent that are 40 or over with highest incidence in USA, Scandinavia, Britain and Australia. 
  • The disease shows cell proliferation, high levels of collagen and extra-cellular matrix remodelling. 
They also make a key point which is what frustrates many of us with the disease and this is that:

"Treatment of DD is not curative" and this means that rather than trying to rid the body of the disease they are just trying to remove the symptoms.

One current treatment that is showing promise is Xiaflex which is a collagenase that digest the triple helical structure of collagen and therefore removal the lumps and whilst this is significantly less invasive than surgery it does have a much higher level of recurrence.

Aim: Their aim is to investigate the functional effects of Xiaflex in comparison to Collagenase A when used on cells cultured from DD nodules, cord, fat and skin. They look into various different aspects of the cells including looking at the cell growth and at gene expression.

So I am going to try and go through this and explain the key points one figure at a time to make it easier to follow.

Figure 1: Here they just show the process through which they have derived the cells and then the experiments that they are planning to do on them.

Figure 2 (and Figure 3): Here they are measuring the ability of the cells from the DD nodules and cords to grow in the presence of  Xiaflex / Collagenase A and whether they are able to recover from this treatment in 24 hours by removing the drug and continuing to monitor the cells. In Figure 3: They also look at fat and skin cells from DD patients in a similar way.

  • They find that without treatment there is no significant different between the nodules and cords compared to the skin and fat although the DD nodules and cords do show a faster growth rate. This makes sense as it is a proliferative disease.
  • Treatment with  Xiaflex / Collagenase A decreases all of the things that they are measuring in ALL cell types in a dose dependent manner. 
  • 24 hours after removal of the drug the cells recover, this happens even at the highest dose and may help to explain why DD comes back so frequently when treated with  Xiaflex
Figure 4: I can't really follow this figure very well so rather than get it wrong and make mistakes I am going to leave this figure out but their key point from this seems to be that Xiaflex / Collagenase A induce membrane leakage and reduce cellular viability and metabolic activity in DD cells.

Figure 5: Here they are looking at the toxicity of the 2 different drugs and want to see which is more toxic to cells and if there is any specificity towards DD nodules and cords. 
  • Xiaflex / Collagenase A both cause cells to die. 
  • Collagenase A causes both necrosis (premature cell death) and apoptosis (programmed cell death) 
  • Xiaflex only causes necrosis.
As far as I can tell from reading this (and correct me if I am wrong) they do not comment on whether this is a big deal or anything? But I think they say in the discussion that it may mean that there are likely to be viable DD cells and therefore there is an increased chance of disease recurrence. 

Figure 6: Here they are looking in the different types of cells at the levels of mRNA for key components in the production of collagen to see if this is altered by the presence of Xiaflex / Collagenase A. For those who don't know about the central dogma of life I have tried to outline it in the diagram below. But the key thing you need to know to understand these results are that mRNA is produced from DNA and that the levels of mRNA vary so that it can be used as an indicator of protein levels in the cell as protein is produced from mRNA. (Below image is VERY simplified)
The central dogma: DNA is where the information is stored and this is processed into RNA and then proteins. 


  • They are looking in DD disease and see that  Xiaflex treatment causes a decrease in levels of collagen I and collagen III as well as fibronectin (forms part of ECM and interacts with collagen), alpha SMA and TGF-B1. If you remember from my previous post a decrease in TGFB1 would be useful. Explained in the image below. 

Central blue boxes show state in DD cells, right hand side is reported influence of Xiapex in DD cells

  • They also say that levels of collagen are at their highest in the DD nodule which is exactly what you would expect. 
Figures 7 & 8: In figures 7 and 8 they are looking at the protein levels of Collagen I and III in the different cells types to see a) is there any difference in their profiles between the different sources (skin, fat, nodule and cord) and b) does Xiaflex / Collagenase A treatment cause any changes to these levels
  • They show that the levels of Collagen I and III in the nodules are very high with Collagen III in particular very, very high (basically two times more than seen in anywhere else). 
  • When looking at the effects of Xiaflex and Collagenase A they see that: 
  • Collagenase A has an influence on Collagen I by causing a decrease on all samples at the highest dose only. 
  •  Collagenase A only decreases Collagen III at the highest 2 doses in the nodules, cords and fat.
  • Xiaflex causes a large decrease on all samples at all doses for both Collagen I & III with the largest decrease observed in the Nodules and cords.  
Figure 9: Here they are trying to show that the decreased levels of Collagen and other ECM factors was not just at the mRNA level but that they were also decreased at the protein level when DD tissue was treated with Xiaflex. 
  • Xiaflex does down regulate the protein levels of fibronectin, Collagen and CTGF (and more) at  the protein level. 
Figure 10: I am just going to say that they see that Xiaflex and Collagenase causes a decrease in cell cycle markers. This makes sense as when the drug is added you see a slowing on cell growth and this means that the cell cycle is slower and therefore you should have a decrease in cell cycle related proteins (though in this case they are look at mRNA).

Right so that is all that I am going to cover for this paper, as I said it is a long post and it is very science based so here is the summary for all those who just want the key points or who are just too lazy to read it all (which is fair enough). 

Summary
  • DD nodules and cords do show a faster growth rate.
  • Xiaflex and collagenase A both decrease the growth rate in DD cells. 
  • Xiaflex treatment causes a decrease in levels of collagen I and collagen III as well as fibronectin  and TGF-B1 (see the diagram as to why this might be good). 
  • Levels of Collagen I and III in the DD nodules.

Reference:
Syed F, Thomas AN, Singh S, Kolluru V, Emeigh Hart SG, et al. (2012) In Vitro Study of Novel Collagenase (XIAFLEX®) on Dupuytren's Disease Fibroblasts Displays Unique Drug Related Properties. PLoS ONE 7(2): e31430. doi:10.1371/journal.pone.0031430