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Identification of novel markers and molecular targets in pathological fibrosis in scleroderma skin lesions

Dr. Pravin Hissaria - Division of Human Immunology, IMVS/SA Pathology

We know very little about how scleroderma develops. This study will look at why cells change their basic characteristics in the disease. This is a completely new approach and, if proven, the aim is to pxploit the regulatory mechanisms of this phenomenon to reverse fibrosis.

 

Dr. Hissaria is the immunology staff specialist at IMVS/South Australia Pathology and Royal Adelaide Hospital. She has received numerous awares and, most recently, was invited as guest speaker at the Royal College of Pathologists (RCPA) annual conference in Sydney.

Scientific Report

Chief Investigators

Pravin Hissaria, Yeesim Khew-Goodall, Susanna Proudman
Division of Human Immunology, Istitute of Medical and Veterinary Science/SA Pathology, Adelaide SA 5000

Original Aims
  1. To test the hypothesis that the miR-200 microRNAs are down-regulated and Pez expression is elevated in scleroderma fibrosis and that these correlate with elevated TGF expression.
  2. To test the hypothesis that other microRNAs are involved in the process of fibrogenesis leading to skin fibrosis in scleroderma.
Results

During the year that the project was funded, we began accruing both scleroderma patient samples and normal control skins but the numbers of SSc patients consenting for skin biopsies have been lower than expected (4 to date) and the process is still ongoing to obtain up to 10 patients. While this process is ongoing, we started collecting and freezing peripheral blood mononuclear cells (PBMCs) and serum from scleroderma patients with plans to extract microRNAs from peripheral blood. This will also help in the validation of any alterations in the microRNA (miR) levels in skin biopsies by comparing them with peripheral blood. To date, we have stored PBMCs from 53 well characterised scleroderma patients which can be used for the above purpose. We have also set up and used an in vitro cell culture system of primary dermal fibroblast treated with TGF to mimic the fibroblast activation observed in skin fibrosis associated with SSc (Fig. 2a).

Using this in vitro cell culture system we have shown:

  1. That the miR-200 family of microRNAs, miR-205 and miR-203 are down-regulated when dermal fibroblasts are exposed to TGF (Fig. 1) (Aim 1).
  2. By microRNA microarray analysis that the expression of a number of other microRNAs are also altered when dermal fibroblasts are exposed to elevated TGF levels (Fig.2b) (Aim 2).

Of particular interest for follow-up analysis are the miR-29 family (miR-29a, b, c) and miR-424 as both these miRs target proteins that are pertinent to the pathology of SSc and indeed all fibrosis. We have verified the changes in miR-29b and miR-424 expression by Taqman real time microRNA qPCR. To overcome donor-specific differences, the qPCRs were performed using fibroblasts explanted from 3 different normal donors (one of which was also used in the array analysis). We found that mir-29b and miR-424 were up- and down-regulated, respectively, by TGF by between 2- to 5-fold for miR-29b and by 4- to 7-fold for miR-424 (Fig. 3), in concordance with the array data.

The targets of miR-29 family include a number of collagens and lysyl oxidases whose expression are increased when the miR-29s are downregulated in fibrosis leading to increased collagen content and cross-linking. We and others have verified that these are bona fide targets (data not shown). An interesting downstream but indirect target of miR-424 that we have identified is semaphorin 7A which has been shown to play a critical role in onset of lung fibrosis (Kang H-R et al. (2007) J Exp Med 204:1083). We have also verified that treatment of dermal fibroblasts with TGF and overexpression of miR-424 to mimic its response to TGF treatment both led to increased sema 7A expression (Fig. 6). The changes in miR-29s and miR-424 and their respective targets will be verified using SSc patient samples when we have collected the minimal number required to obtain statistical significance.

Most importantly, these findings formed the groundwork and preliminary data for a successful NHMRC project grant application (ID 626945) to commence in 2010.

Lay report

During the year that the project was funded, we began accruing both scleroderma patient samples and normal control skins but the numbers of SSc patients with early aggressive disease and consenting for skin biopsies have been lower than expected (4 to date) and the process to obtain samples from up to 10 patients is still ongoing. In the meantime, we have started storing immune cells from the peripheral blood of SSc patients which can also be used to test our hypothesis of abnormalities in certain genetic elements (micro RNAs) in patients with this disease. We also set up an experimental model of this condition by using normal skin samples (obtained from consented patients undergoing plastic surgeries for abdominoplasty and breast reductions) to test our hypothesis. Results from experiments run on three normal skin samples yielded encouraging results and were consistent with our hypothesis that abnormalities in genetic elements (micro RNAs) can lead to changes with characteristics of scleroderma in the skin cells. Most importantly, these findings have been useful as a pilot project in giving credence to our hypothesis and have helped us secure a NHMRC project grant (application ID 626945) to commence in 2010. Further work is being undertaken to validate these findings in scleroderma skin biopsies and peripheral blood samples and looking at ways to manipulate this system to halt progression of fibrosis.