Background: Pulmonary arterial hypertension (PAH) is a major cause of death in systemic sclerosis (SSc). Early detection may improve patient outcomes. Methods: We searched for circulating miRNAs that would constitute biomarkers in SSc patients with PAH (SSc-PAH). We compared miRNA levels and laboratory parameters while evaluating miRNA levels in white blood cells (WBCs) and myofibroblasts. Results: Our study found: 1) miR-26 and miR-let-7d levels were significantly lower in SSc-PAH (n = 12) versus SSc without PAH (SSc-noPAH) patients (n = 25); 2) a positive correlation between miR-26 and miR-let-7d and complement-C3; 3) GO-annotations of genes that are miR-26/miR-let-7d targets and that are expressed in myofibroblast cells, suggesting that these miRNAs regulate the TGF-β-pathway; 4) reduced levels of both miRNAs accompanied fibroblast differentiation to myofibroblasts, while macitentan (endothelin receptor-antagonist) increased the levels. WBCs of SSc-noPAH and SSc-PAH patients contained equal amounts of miR-26/miR-let-7d. During the study, an echocardiograph that predicted PAH development, showed increased pulmonary artery pressure in three SSc-noPAH patients. At study initiation, those patients and an additional SSc-noPAH patient, who eventually developed PAH, had miR-let-7d/miR-26 levels similar to those of SSc-PAH patients. This implies that reduced miR-let-7d/miR-26 levels might be an early indication of PAH. Conclusions: miR-26 and miR-let-7d may be serological markers for SSc-PAH. The results of our study suggest their involvement in myofibroblast differentiation and complement pathway activation, both of which are active in PAH development.

Systemic sclerosis (SSc) is an autoimmune disease characterized by fibrosis of the skin and internal organs. Key players mediating fibrosis are myofibroblasts (MF) that, following transforming growth factor β (TGFβ) exposure, produce a collagen-rich extracellular matrix (ECM) that induces myofibroblast differentiation. Myofibroblasts express αvβ3 integrin (a membrane receptor for thyroid hormones) and miRNA-21 that promotes deiodinase-type-3 expression (D3), causing the degradation of triiodothyronine (T3) that attenuates fibrosis. We hypothesized that αvβ3 affects the fibrotic processes through its thyroid hormones (THs) binding site. To test this, dermal fibroblasts (DF) were cultured with/without TGFβ and removed with a base, leaving only normal/fibrotic ECMs in wells. Then, DF were cultured on the ECMs with/without tetrac (αvβ3 ligand, T4 antagonist), and evaluated for pro-fibrotic characteristics, αvβ3, miRNA-21, and D3 levels. Blood free-T3 (fT3), miRNA-21 levels, and the modified Rodnan skin score (MRSS) were evaluated in SSc patients. We found that the “fibrotic-ECM” significantly increased the pro-fibrotic characteristics of DF and the levels of miRNA-21, D3, and αvβ3, compared to the “normal-ECM.” Tetrac significantly inhibited the effects of the “fibrotic-ECM” on the cells. In accordance with tetrac’s effect on D3/miRNA-21, a negative correlation was found between the patients’ fT3 to miRNA-21 levels, and to the development of pulmonary arterial hypertension (PAH). We conclude that occupying the THs binding site of αvβ3 may delay the development of fibrosis.

BackgroundSystemic sclerosis (SSc) is an autoimmune disease characterized by tissue fibrosis of the skin and internal organs. Key players in SSc are myofibroblast cells (MF) that produce large amounts of extracellular matrix (ECM), which facilitates fibrosis. TGFβ is a key factor involved in MF differentiation and collagen-rich ECM (fibrotic-ECM) secretion. Most of the TGFβ is bound to the ECM through latency-associated peptide (LAP) that contains the amino acid sequence arginine-glycine-asparagine (RGD motif). In order to be active, TGFβ must be released from the ECM by proteases like MMP9 or αv integrins that bind LAP through their RGD recognition site. MF express high levels of the integrin αvβ3. Interestingly, αvβ3 is a special integrin since it also serves as a membrane thyroid hormone (TH) receptor that binds T3 and especially T4. The TH binding sites are located proximal to the RGD binding site. MF also express high levels of the pro-fibrotic miRNA-21, which regulates the TGFβ pathway and the level of DIO3 enzyme, which degrades T3. T3 attenuates fibrotic processes. Since high levels of αvβ3 integrin are expressed by MF in the fibrotic sites and the TH binding site of αvβ3 is close to the RGD recognition site, we hypothesized that the TH binding site of αvβ3 is involved in mediating the fibrotic process.Objectives1. To examine the effect of fibrotic-ECM produced by dermal fibroblasts (DF) triggered by TGFβ, on naive DF phenotype and their αvβ3 expression; 2. To examine the connection between the thyroid hormone binding site of αvβ3 integrin to fibrosis-related processes.MethodsDF were isolated from healthy skin, cultured without/with TGFβ, and removed by NH4OH, leaving the normal/ fibrotic ECMs in the wells. ECMs were evaluated for collagen-I and TGFβ levels (Immunohistochemistry, western blot). The effect of the normal/fibrotic ECMs on the following parameters of naïve DF was evaluated: phenotype; expression of MF biomarkers (collagen, elastin and αSMA), expression of αvβ3 integrin, miRNA-21, and DIO3 enzyme; activation of the TGFβ pathway (Smad3 phosphorylation); and MMP9 activity (automatic cell counter, western-blot, RT-qPCR, gelatin zymography). Moreover, the effect of tetraiodothyroacetic acid (tetrac, T4 derivative, and αvβ3 ligand) on the fibrotic-ECM ability to induce DF differentiation into MF and to express αvβ3, miRNA-21 and DIO3 was studied. Last, free T3 levels were measured in 18 SSc patients of whom 7 had pulmonary arterial hypertension (PAH). The patients were divided according to high (n=6)/low (n=12) T3 levels into 2 groups and the difference in circulating miRNA-21 levels between the groups was evaluated.ResultsFibrotic-ECMs generated by TGFβ-triggered DF had higher levels of collagen-I and TGFβ in comparison to normal ECM. In comparison to the normal ECM, the fibrotic-ECM increased the followings in the DF: 1) ability to liberate TGFβ from the ECM by increasing MMP-9 activity and αv expression. 2) TGFβ activation and differentiation to MF evidenced by increased expression of phosphorylated Smad3, αSMA, collagen, and elastin, and increased proliferation. 3) Expression of αvβ3, miRNA-21 and DIO3 (all changes in 1-3: 24-123%↑, p≤0.05). The addition of tetrac to DF cultured on fibrotic-ECM downregulated cell proliferation, collagen, elastin and aSMA expression to the levels found in DF cultured on normal ECM. Furthermore, it reduced αvβ3, miRNA-21, and DIO3 levels. Accordingly, decreased miRNA-21 levels were found in the high T3 SSc group, compared to the low T3 group that contained all the PAH patients (p≤0.05).ConclusionOur results demonstrated the existence of a vicious cycle: TGF-triggered DF produced a fibrotic-ECM that promoted bystanders’ DF differentiation to MF. Using this model, we demonstrated that tetrac binding to αvβ3 integrin inhibited the differentiation of DF to MF, and their ability to activate the αvβ3/miRNA-21/DIO3/T3 pathway. These results suggest that the thyroid hormone binding site of αvβ3 may be a potential target for the treatment of fibrosis.REFERENCES:NIL.Acknowledgements:NIL.Disclosure of InterestsNone Declared.