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Notes for Delphine Gomes, Ph.D. – U698 Paris 7, aortic smooth muscle cells and epigenetic reprogramming
Aneuryms of ascending (outflow tract, TAA) or descending aorta – inflammatory atherothrombosis.
ECM rich in fibronectin, fibrillin and elastic fibers around the smooth muscle fibers. During the aneurysm, rarification of SMC then elastic and collagen fibers, accumulation of mucopolysaccharides. (Via plasmin and MMPs – activated in TAA).
Already implicated – FBN1, EFEMP2 for matrix; TGFBR1, SLC2A10, TGFBR2 (Marfan and Loeys-Dietz syndromes); ACTA2 and MYH11 are contractile proteins.
Other aneurysms due to bicuspid aortic valve.
For signaling TGFb1 – ocular, skeletal, ligament, CV effects in Marfan and LD syndromes.
The SMC makes TGFb1 in a latent form, assoc with LTBP1, retained in ECM by FBN1 and FN. Proteases liberate the active form that can then cause intracellular signaling, leading to targets among others: alpha-actin (smooth muscle) and CTGF.
TGFBR1/2 act through SMAD2/3-P/4 but also through MAPK/ERK/p38 and RhoA.
The idea for Marfan via FBN1 mutations is that there is TOO MUCH TGFb1 released into ECM in active form, leading to SMAD2-P relocalizing to nucleus (this much is true) and a target is indeed transcribed, CTGF. But this does not necessarily lead to degradation of ECM.
n=15 BAV, n=15 healthy aortas, n=19 degenerative forms, n=15 FBN1 mutes, n=2 TGFB2 mutants.
More latent TGFb1 in the media of the diseased re: normal aorta, also more expn of LTBP1 – yet no more activation or transcription of TGFb1 itself. Co localizes with accumulations of FN and FBN1, degradation of elastic fibers. However, homogenous distribution of Smad2-P in nucleus throughout the whole media of the aorta.
Article in press 2010 at Cardiovascular Research Gomez D et al. Constitutive P of Smad2, not Smad3, in the SMC of the diseased aortae. If add TGFb to cultures of SMC in disease or normal aorta, it will stimulate expression of CTGF (via Smad pathway), but not for the alpha-actine, which is a target of the MAPK pathway.
Overexpression of mRNA in all aneurysms in tissues or SMC in vitro – for Smad2, not 3/4/7. Two alternative first exons for Smad2, three transcripts one of which has BOTH first exons. Overexpn is only for the longer isoforms with the 1a first exon, so the promoter control is relative to that particular exon, not for the more 3' one.
SMCs over expressing SMAD2 and thereby CTGF continue to do so throughout passages in vitro. This is not true for the overexpn of LTBP1 observed in situ and in the early SMC, but is thereby lost.
Epigenetic regulation of SMAD2 overexpression? Cytosine methylation, or posttranslational modifications of histones? Hypothesis was first at level of methylation, so it was not at DNA level, but what other possibilities for histones?
N-terminal domains of H1-H4 are subject to acetylation (activating on lysines) or methylation (activating or inhibitory). Also phosphorylation and ubiquitination are possible.
She managed to do ChIP on frozen human tissue. Nice explanation of ChIP – looking at SMAD2 promoter, did a priori PCR. Results expressed in terms of IP/Input (showed end-point PCR bands rather than qPCR).
H3K9/14ac et H3K4me are activating, and they are indeed enriched. Looked at differences between healthy and diseased TAA aortas. Hyperacetylation of H3 and (less) for Me, at -500 to -400 upstream of first promoter, relative to other amplicons spaced throughout the entire promoter region. Also looked at histone coverage with anti-H3 (non-modified). Somewhat more activity of GCN4 and PCAF, H3 HATs. Looked at transcription of SMAD2 1a and 1b in response to specific H3 HAT inhibitors, and it was affected. Inhibitors are Garcinol and CPTH2.
Which TFs act on this location? p53 and MYC, associated with H3 HATs PCAF and GCN5 to also be acetylated, not just on histones. TRRAP is a co-factor for this assemblage. There are consensus sites from 0 to 400 upstream of 5' promoter for p53 and MYC.
ChIP on SMAD2 promoter also showed enrichment around binding site of p53 that is predicted. However, in the TAA, there was a LACK of recruitment of MYC in this position, usually occupied in the healthy aortas. Very nice mirror image plot along the promoter sequence.
TAA has augmented acetylation of p53 by immunoprecipitation, this is done by PCAF and GCN5 presumably.
Epigenetic regulation effect on SMC via this hyperactivation of SMAD2-P? Hypothesis is that the expression of matrix proteins and serpins (anti-proteases) are upregulated in response – they are constitutively overexpressed in the cultured SMC, PAI and PN1 also have promoters occupied (as shown by ChIP)
SMC with plasmin and plasminogen on healthy cells – add TGFb1, protects against proteolysis and apoptosis. For the TAA, less endogenous plasmin activity (?), and if one adds in an anti-PN1 antibody and protects from proteolysis and apoptosis. So the activation of SMAD2 is supposedly beneficial to the TAA – it is a good or bad global effect?
Protection against addition of plasmin in vitro for effect of apoptosis, but in Marfan mice, get worsening of pathology, perhaps by making the reaction to a punctual aggression something autonomous in the responding SMC.
Interest in the phosphorylation of SMAD2 – not just TGFBR1/2 but also MPS1 can P SMAD2.