Summary of RNA sequencing reveals a depletion of collagen targeting microRNAs in Dupuytren’s disease

http://www.biomedcentral.com/content/pdf/s12920-015-0135-8.pdf

Summary of RNA sequencing reveals a depletion of collagen targeting microRNAs in Dupuytren’s disease

As far as I can tell nothing ground-breaking but interesting nonetheless and potentially it could be ground-breaking in the long run. I will not go into the basics of the science as I have done that many times before but we all have RNA which doesn't actually code for proteins (miRNA) what this does do though is interact with other RNA so that it can't interact with RIbosomes and be translated into a protein. So miRNA can have a massive influence on the levels of proteins within a cell. Proteins are what actually drive the majority of changes in the cells and therefore tissues and therefore the body so the fact that miRNA can impact on the level of protein is very important. 

From what I can make out they are saying that there is a difference in the expression profiles between normal cells and Dupuytren's cells and that in normal cells you see a higher amount of miRNA that target Collagen and ECM proteins etc. It is perhaps the case that lower levels of these in diseased tissue is the cause of the condition or at least part of the effect. They appear to be saying that if you could boost these RNAs in patients then it could be a treatment of sorts and if you can detect them then it could be used as a biomarker to guide where diseased tissue is and determine what needs to be removed during surgery. 

Interestingly in their summary they do not draw attention to the fact that there was more up-regulation in DD tissue than there was in the normal tissue (70 odd miRNA's compared to 30 or so). Check out page 8 for a breakdown of these but the conclusion I am drawing is that the important functional effect is the miRNAs that I mentioned above, the comparatively elevated levels of miRNAs that target Collagen etc in the normal tissue. 

It would be interesting to see if they could detect a difference between patients at different stages, these patients were all advanced cases that were having surgery and perhaps there is a development of the profile over time or indeed a pre-Dupuytren's profile which can reveal which patients are at risk. After all if it is in your genes then it will probably be in your profile. 

"The microRNAs characterized in this investigation have the potential to serve as disease biomarkers that can help guide surgical management by determining optimal surgical margins during open fasciectomy. Novel RNA-therapeutics that are currently in development, also have the potential to target disease specific microRNAs and prophylactically prevent disabling fibrosis minimizing the need for invasive surgical treatments. Fibrosis related microRNAs may also play important regulatory roles in other disorders of fibrosis including scleroderma, idiopathic pulmonary fibrosis, as well as scarring and wound healing."

This is some interesting research and I look forward to hopefully seeing this progress in the future. 

Calorie Restriction to help Dupuytren's and Ledderhose?

A literature review to analyse the link between calorie restriction and pathways associated with Dupuytren's disease

Over recent weeks I have heard several patients talking about calorie restriction and how eating a low calorie diet really seems to help with the pain that they were experiencing with Dupuytren's and Ledderhose and that when they stopped the diet it seemed to get worse. 

The IGF-2 link:

There is some logic behind this, after all I have already discussed how these conditions can be linked to IGF2 and IGF1 is linked to calorie restriction. However further research did not seem to come up with any evidence to suggest a good link between IGF1 and collagen. The only way I could see that the IGF2 information could help is as follows: 

• I know that IGFBP6 binds to both IGF1 and IGF2, albeit more strongly to IGF2, and that calorie restriction causes a decrease in IGF1.

• So of you have less IGF1 then the IGFBP6 is more likely to bind to IGF2, assuming that the concentrations are at a level where the increased availability of IGFBP2 still results in an increased binding affinity for IGF2. 

• So if low IGF1 causes an increase in IGFBP6 binding to IGF2 and therefore decrease the downstream effects of IGF2 one of which is collagen production you may see a difference http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3684083/figure/F3/

I think that the above is a little bit of a stretch but you never know. That was all I could find using IGF1 and collagen.

Figure 1: Overview of the impact of TGF-B1 on Collagen production and how calorie restriction impacting IGF-1 levels may result in a lowering of Collagen production.

Calorie Restriction and Collagen:

So instead of that I took a different approach and decided to look for a straight linked between calorie restriction and collagen. Here I managed to find a link, in that the following paper states that calorie restriction results in a decrease in collagen production, perhaps this would also hold true a) in humans b) under conditions where Dupuytren's is present. (Ref 1). I did not have full access to the paper so could not really see what they had done but it also states that this decreased collagen production also has a negative impact on would healing which of course relies, to some extent, on the same pathways as Dupuytren's. This may be a good thing for Dupuytren's or Ledderhose patients.  

The MMPs: 

Another key set of proteins in the extracellular matrix (ECM) is the MMP protein. These are involved in breaking things down and it has been shown that knock-down (removal in cells) of  MMP2 (Ref 2) inhibited cell mediated contraction. Interestingly there have been studies in rats that have shown that a calorierestricted diet results in a decrease in MMP2, so you could argue that a calorie restriction diet will lower MMP2 and that a lowered MMP2 will aid Dupuytren's and or Ledderhose. Furthermore they showed that it appears that calorie restriction also lowers the TGFB1 pathway which I discuss in the IGF2 link at the top. 

Figure 2: The role of MMP2 in Dupuytren's and the potential impact of calorie restriction on Dupuytren's and Collagen production. 

Collagen, the extra-cellular matrix and calorie restriction:

My final search I just thought I would have a look for ECM itself and calorie restriction and see what I could find and I found a couple of very interesting papers which I believe are both free to download. 

The first paper (Reference 4) is looking at the impact of calorie restriction in tumours. Interestingly they found that there was a decrease, as expected in IGF-1 which I have discussed above but they also observed that in calorie restricted mice there was a decrease in MMP2, which as discussed above is linked to the development of Dupuytren's. There was also a decrease in the levels of TGFB-1 which again I have also discussed above. 

Another protein that they mentioned that took my interested was collagen 4. Although Collagen 3 is the main component of the Dupuytren's lumps it is interesting that there are downstream effects on the levels of a collagen type. The second paper I am not going to go into as it basically has similar but less information as in the above paper though it does have a good picture on page 6 (Reference 5). 

The other side of the argument: 

You cannot construct a complete article without looking at both sides of the story. My research into this side was not as extensive because it is hard to find information disproving something that it just an idea but did bring up some interesting points.

The following paper (Ref 6) shows that despite the information I have seen above that TGF-B2 (yes 2 not 1, still looking into this but from what I have seen so far 1 and 2 act through the same pathways) does not affect the collagen levels in Dupuytren's cultures. In fact I am not sure what else to say on this side, if anyone has any information they would like to add then let me know.

Conclusions: 

Would having a calorie restricted diet hurt someone? It might but at the same time you can come off of it and may not have lost anything but you may have gained the used of your foot or stopped your hand from progressing. There is no information out there that says that it will work like that, in fact there is no direct evidence at all linking calorie restriction and Dupuytren's (for or against). The above data is a collection of the information that I could find that linked calorie restriction to pathways that have been linked to Dupuytren's. A lot of this data suggests that there could be some impact but there was nothing there that made me think that it definitely will work. 

As a non-medical professional I cannot endorse having a low calorie diet in general or a as a treatment for these conditions, although the information above suggests that it may have some impact. Note that many of the above studies were in cells or cancer and not in humans and none were related directly to Dupuytren's or related conditions. You should always consult a doctor before starting a very low calorie diet. If a doctor was interested in using this then I would be happy to work with them to look into this.

If any patients have experience of this and would like to share their story it would be great to add the information to the blog.

Other areas of interest: 

There is another signalling pathway called the Wnt signalling pathway. I know from back in my degree that this is using is development and it is still active in adults. I mention this pathway because it has been shown that a high number of Dupuytren's patients have SNPs (differences to everyone else) in genes that are associated with that patients (Ref 7). Although this is as far as the evidence go the Wnt signalling pathway has been linked to diabetes, cell growth and certainly warrants more investigation. 

Reference 1:

Reiser K1, McGee C, Rucker R, McDonald R - 

Access 29-09-2014

J Gerontol A Biol Sci Med Sci. 1995 Jan;50A(1):B40-7.Effects of aging and caloric restriction on extracellular matrix biosynthesis in a model of injury repair in rats.

http://www.ncbi.nlm.nih.gov/pubmed/7814778

Reference 2: 

Biochim Biophys Acta. 2012 Jun;1822(6):897-905. doi: 10.1016/j.bbadis.2012.02.001. Epub 2012 Feb 9. MMP-14 and MMP-2 are key metalloproteases in Dupuytren's disease fibroblast-mediated contraction.

Wilkinson JM1, Davidson RK, Swingler TE, Jones ER, Corps AN, Johnston P, Riley GP, Chojnowski AJ, Clark IM.

http://www.ncbi.nlm.nih.gov/pubmed/22342364

Reference 3: 

Calorie Restriction Reduces MMP-2 Activity and Retards Age-associated Aortic Restructuring in Rats , Mingyi Wang et al

http://circ.ahajournals.org/cgi/content/meeting_abstract/114/18_MeetingAbstracts/II_335-b

Reference 4:

Caloric restriction reduces growth of mammary tumors and metastases - Mariana S. De Lorenzo et al

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3165123/

Reference 5: 

Molecular Mechanisms of Calorie Restriction’s Protection against Age-related Sclerosis, Elena Chiarpotto et al - http://onlinelibrary.wiley.com/doi/10.1080/15216540601106365/pdf

Reference 6: 

Enhanced Dupuytren's disease fibroblast populated collagen lattice contraction is independent of endogenous active TGF-β₂ Raymond Tse¹⁴, Jeffrey Howard¹²⁴, Yan Wu¹⁴ and Bing Siang Gan^1234*

http://www.biomedcentral.com/1471-2474/5/41

Reference 7: 

Wnt Signaling and Dupuytren's Disease, Guido H. Dolmans,  et al

http://www.nejm.org/doi/full/10.1056/NEJMoa1101029

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