1. Scleroderma ( Systemic Sclerosis ) and Aquaporins Presented by: Bahman yousefi Supervisor: Dr. mahdi mahmoudi

2. Definition 1. Systemic sclerosis (scleroderma) a multisystem disorder characterized by 1) Functional and structural abnormalities of blood vessels 2) Fibrosis of the skin and internal organs 3) Immune system activation 4) Autoimmunity 1. Systemic sclerosis (scleroderma) a multisystem disorder characterized by 1) Functional and structural abnormalities of blood vessels 2) Fibrosis of the skin and internal organs 3) Immune system activation 4) Autoimmunity

3. Epidemiology 1.Prevalence: 4-12 new cases per million per year 2. Susceptibility: host factor 1) age - peak occurrence: age 35-65 years 2) gender - female : male = 3-12 : 1 3) genetic background 1.Prevalence: 4-12 new cases per million per year 2. Susceptibility: host factor 1) age - peak occurrence: age 35-65 years 2) gender - female : male = 3-12 : 1 3) genetic background

4. Classification 1.Systemic sclerosis –Diffuse cutaneous systemic sclerosis –Limited cutaneous systemic sclerosis –Overlap syndromes 2. Localized scleroderma –Morphoea –Linear scleroderma 1.Systemic sclerosis –Diffuse cutaneous systemic sclerosis –Limited cutaneous systemic sclerosis –Overlap syndromes 2. Localized scleroderma –Morphoea –Linear scleroderma

5. Etiology Environmental factors 1) Silica Dust 2) Organic Solvents 3) Biogenic Amines 4) Urea Formaldehyde 5) Polyvinyl Chloride 6) Rapeseed Oil 7) Bleomycin 8) L-tryptophan 9) Silicone Implant (?) Environmental factors 1) Silica Dust 2) Organic Solvents 3) Biogenic Amines 4) Urea Formaldehyde 5) Polyvinyl Chloride 6) Rapeseed Oil 7) Bleomycin 8) L-tryptophan 9) Silicone Implant (?) Genetic predisposition Defective immunoregulation 1) cell mediated immunity CD4/CD8, cytokines 2) Humoral immunity – Hypergammaglobulinemia – Autoantibody Production – Antinuclear Antibody (+) > 95%

6. Clinical features 1.Vascular abnormalities 1) Raynaud's phenomenon - cold hands and feet with reversible skin color change (white to blue to red) - induced by cold temperature or emotional stress - initial complaint in 3/4 of patients - 90% in patients with skin change 1.Vascular abnormalities 1) Raynaud's phenomenon - cold hands and feet with reversible skin color change (white to blue to red) - induced by cold temperature or emotional stress - initial complaint in 3/4 of patients - 90% in patients with skin change

7. Telangiectasia Local disruption of angiogenesis Clinical features

8. 2. Skin involvement (1) 1) stage 2. Skin involvement (1) 1) stage Edematous Phase Indurative Phase Atrophic Phase 3) Decrease in range of motion, loss of facial expression, inability to open mouth fully, contractures

9. Facial changes Tight, thin lips with vertical perioral furrows

10. Clinical features 2. Skin involvement (2) 2. Skin involvement (2) ulceration, loss of soft tissue of finger tip, pigmentation, calcific deposit, capillary change 3. Musculoskeletal system Polyarthritis and flexion contracture Muscle weakness and atrophy (primary /secondary)

11. Clinical features 4. Intestinal Involvement 1) Esophagus: hypomotility and retrosternal pain, reflux esophagitis, stricture 2) Stomach: delayed emptying 3) small intestine: pseudo-obstruction, paralytic ileus, malabsorption, weight loss, cachexia 4) Large intestine: chronic constipation and fecal impaction diverticula 4. Intestinal Involvement 1) Esophagus: hypomotility and retrosternal pain, reflux esophagitis, stricture 2) Stomach: delayed emptying 3) small intestine: pseudo-obstruction, paralytic ileus, malabsorption, weight loss, cachexia 4) Large intestine: chronic constipation and fecal impaction diverticula

12. Clinical features 5. lungs 1) 2/3 of patients affected - leading cause of mortality and morbidity in later stage of systemic sclerosis 2) Pathology - interstitial fibrosis - intimal thickening of pulmonary arterioles (pulmonary hypertension) 3) Complains - dry cough, breathlessness 5. lungs 1) 2/3 of patients affected - leading cause of mortality and morbidity in later stage of systemic sclerosis 2) Pathology - interstitial fibrosis - intimal thickening of pulmonary arterioles (pulmonary hypertension) 3) Complains - dry cough, breathlessness

13. Pulmonary fibrosis

14. Clinical features 7. kidney 1 ) Diffuse scleroderma in association 2) pathology - intimal hyperplasia of the interlobular artery - fibrinoid necrosis of afferent arterioles - glomerulosclerosis 3) Proteinuria, abnormal sediment, azotemia, microangiopathic hemolytic anemia, renal failure 7. kidney 1 ) Diffuse scleroderma in association 2) pathology - intimal hyperplasia of the interlobular artery - fibrinoid necrosis of afferent arterioles - glomerulosclerosis 3) Proteinuria, abnormal sediment, azotemia, microangiopathic hemolytic anemia, renal failure

15. Clinical features 6. Heart (10%) 1) Pericarditis 2) Heart Failure 3) Arrhythmia 4) Myocardial Fibrosis 6. Heart (10%) 1) Pericarditis 2) Heart Failure 3) Arrhythmia 4) Myocardial Fibrosis

16. Pathogenesis

17. The initial events in SSc Vascular Damage 2- Fibroblast activation Initiate microvascular damage A- Leakage of substances B- Capable of producing further EC damage C- progressive disorganization of the vascular architecture 3- Gelatinase activity of fibroblasts 1- Target of an inflammatory cell infiltration Pathogenesis

18. Fibroblasts & Fibrosis Fibroblasts maintain the structural integrity of connective tissue - Secreting fibrillar procollagens, fibronectin -Regulating the turnover and composition of the ECM via highly specific Proteases such as collagenase Fibroblasts appear to orchestrate the 1- Production 2- Deposition 3- Remodeling of collagens and other ECM Fibroblasts maintain the structural integrity of connective tissue - Secreting fibrillar procollagens, fibronectin -Regulating the turnover and composition of the ECM via highly specific Proteases such as collagenase Fibroblasts appear to orchestrate the 1- Production 2- Deposition 3- Remodeling of collagens and other ECM Pathogenesis

19. Quiescent fibroblasts can be activated by different mechanism 1)Direct cell–cell contact 2) Stimulation by soluble mediators TGF-b, connective tissue growth factor (CTGF), PDGF 3) Modulation of cell–matrix interactions 4) Altered gene expression in SSc fibroblasts Quiescent fibroblasts can be activated by different mechanism 1)Direct cell–cell contact 2) Stimulation by soluble mediators TGF-b, connective tissue growth factor (CTGF), PDGF 3) Modulation of cell–matrix interactions 4) Altered gene expression in SSc fibroblasts Fibroblasts & Fibrosis

20. SSc may result in preferential selection and/or activation of high collagen- producing subpopulations of fibroblasts a- Decreased susceptibility of some fibroblasts to apoptotic cell death b- Altered cell turnover SSc may result in preferential selection and/or activation of high collagen- producing subpopulations of fibroblasts a- Decreased susceptibility of some fibroblasts to apoptotic cell death b- Altered cell turnover Pathogenesis Fibroblast Apoptosis Apoptosis of fibroblasts,terminating fibroproliferative responses induced by tissue injury after resolution of the inflammatory phase Fibroblasts are activated and overproduce ECM proteins, such as collagens I, III, V, VI and VII, tenascin, proteoglycans, fibronectin, laminin and fibrillin-1

21. TGF-b and cytokines involved in the pathogenesis of SSc -TGF-b plays an important role in the initiation and development of fibrosis - CTGF is mainly responsible for the maintenance of fibrotic lesions TGF-b signaling, which involves Smad proteins, is altered in SSc fibroblasts 1- Decreased expression of inhibitory Smad 7 2- Increased expression of coactivator Smad 3 -TGF-b plays an important role in the initiation and development of fibrosis - CTGF is mainly responsible for the maintenance of fibrotic lesions TGF-b signaling, which involves Smad proteins, is altered in SSc fibroblasts 1- Decreased expression of inhibitory Smad 7 2- Increased expression of coactivator Smad 3

22. TGF-b and cytokines involved in the pathogenesis of SSc

23. Autoantibodies  Specific autoantibodies -Anti-topoisomerase 1 Ab (ATA), 30–70% of patients with diffuse SSc, -Anti-centromere Ab (ACA), detected in 22–40% of SSc patients and more often in those with limited disease -Anti- RNAP Ab, found in 4–33% of diffuse SSc patients, in association with renal involvement and represent a marker of poor prognosis.  Non-specific autoantibodies  Specific autoantibodies -Anti-topoisomerase 1 Ab (ATA), 30–70% of patients with diffuse SSc, -Anti-centromere Ab (ACA), detected in 22–40% of SSc patients and more often in those with limited disease -Anti- RNAP Ab, found in 4–33% of diffuse SSc patients, in association with renal involvement and represent a marker of poor prognosis.  Non-specific autoantibodies Pathogenesis

24. Autoantibodies Pathogenesis

25. Extracellular-Matrix Components and Their Receptors  ECM modulate cellular responses by 1- Regulating the activity of cytokines 2- Growth factors  ECM also provides points of adhesion -Mediating outside-in and inside-out signaling The macromolecular arrangement of collagens in scleroderma is altered Integrin α1β1 - Elicits signals to down-regulate collagen synthesis by fibroblasts - Fibroblasts in SSC have reduced surface levels of α1β1 integrin, resulting amplify fibrosis in scleroderma

27. Susceptible people Environmental factors Activation of the immune system Endothelial cell activation / injury Fibroblast activation The final stages of the Pathogenesis - High Production of collagen- main complications - Abnormal secretion of ECM Autoantibody production Cause skin hard, thick and also lead to deformity in some parts of the body including the face, mouth and hands Pathogenesis Endothelial cell activation / injury Fibroblast activation Autoantibody production

28. Aquaporin and SSc Aquaporin and SSc

29. Aquaporin topology and structure  AQP, (Preston et al., 1992), -were identified,water channel, aquaporin -small 30kd,hydrophobic, monomernes  Currently 13 members AQP0- AQP12, identified in mammals

30. AQP - A subset of the AQPs, the aquaglyceroporins AQP3, AQP7 and AQP9, transport both water and glycerol - AQP9 can also transport other small polar solutes, including amino acids, sugars and even arsenite - AQP6 has been proposed to function as an intracellular chloride channel - The functions and cellular localization of AQPs 10–13 are not clear - A subset of the AQPs, the aquaglyceroporins AQP3, AQP7 and AQP9, transport both water and glycerol - AQP9 can also transport other small polar solutes, including amino acids, sugars and even arsenite - AQP6 has been proposed to function as an intracellular chloride channel - The functions and cellular localization of AQPs 10–13 are not clear

31. Water-selective aquaporins

36. AQP3 in skin hydration AQP3-facilitated glycerol transport in skin Important determinant of epidermal and stratum corneum hydration AQP3-facilitated glycerol transport in skin Important determinant of epidermal and stratum corneum hydration Aquaglyceroporins (water and glycerol transport)

37. AQP3 in cell proliferation Aquaglyceroporins (water and glycerol transport)

39.  The ERK pathway is involved in hEGF-induced AQP3 expression, cell migration and proliferation in the human gastric cancer cell lines  Chao and colleagues in a study in 2010 found that EGF via EGFR, PI3K و ERK  The ERK pathway is involved in hEGF-induced AQP3 expression, cell migration and proliferation in the human gastric cancer cell lines  Chao and colleagues in a study in 2010 found that EGF via EGFR, PI3K و ERK  In normal skin fibroblasts and keratinocytes, EGF AQP3  Tokiyama et al, in 1990 on 3 scleroderma patients, affinity of EGFR to EGF, it seems for this reason inadequate expression of AQP3 AQP3, increased cell migration

40.  Matrix metalloproteinases (MMPs) in key regulators of the formation & degradation of ECM  According to the above. In relation to the skin dryness & Delay in wound healing It seems skin fibroblast cells of SSC patient, AQP3 levels are lower than normal  Matrix metalloproteinases (MMPs) in key regulators of the formation & degradation of ECM  According to the above. In relation to the skin dryness & Delay in wound healing It seems skin fibroblast cells of SSC patient, AQP3 levels are lower than normal The Role of the AQP3 in fat synthesis, fluid transport Cell proliferation, Fibroblast migration in damaged areas Synthesis & secretion of MMPs (collagenase )  It is Possible Inadequate Stimulation, Defects in EGF Receptor, Signaling Failure or Defect in the AQP3 gene Physiological Pathological  Hao and et al study,turned off the AQP3 gene Expression of MMPs and expression of AQP3 Expression of MMPs

41. The main objectives 1.Evaluation of AQP3 gene expression in protein and RNA levels in fibroblasts of SSC patients 2.Determine effective influencing Mechanisms in AQP3 gene expression in fibroblasts of patients SSC 1.Evaluation of AQP3 gene expression in protein and RNA levels in fibroblasts of SSC patients 2.Determine effective influencing Mechanisms in AQP3 gene expression in fibroblasts of patients SSC Secondary objectives  Determine AQP3 gene expression in mRNA and protein levels  Determine amount of EGF protein  Determine amount of p-EGFR and EGFR proteins  Determine amount of ERK and p-ERK proteins  Determine the levels of PI3K protein  Determine the effect of EGF on the AQP3 expression in the presence of TGF-β in skin fibroblasts of SSC patients compared with healthy control

42. Practical Objectives  This research can identify - Role of the AQP3 pathogenicity of systemic sclerosis disease -Also identify possible therapies based on functions of this protein -And effective mechanisms of this protein expression levels in future -  This research can identify - Role of the AQP3 pathogenicity of systemic sclerosis disease -Also identify possible therapies based on functions of this protein -And effective mechanisms of this protein expression levels in future -